Protocol development for reliable isolation of tear-film ...

Protocol development for reliable isolation

of tear-film neutrophils and in vitro

functionality testing.

by

Yutong Jin

A thesis

presented to the University of Waterloo

in fulfillment of the

thesis requirement for the degree of

Master of Science

in

Vision Science and Optometry

Waterloo, Ontario, Canada, 2019

©Yutong Jin 2019

ii

AUTHOR'S DECLARATION

I hereby declare that I am the sole author of this thesis. This is a true copy of the

thesis, including any required final revisions, as accepted by my examiners.

I understand that my thesis may be made electronically available to the public.

iii

Abstract

During prolonged eye closure, such as during sleep, leukocytes are recruited to the

ocular surface, with polymorphonuclear neutrophils (PMNs) representing the largest

population.

PMNs are essential inflammatory cells of the innate immune system, and possess

several efficient killing mechanisms, such as phagocytosis and release of antimicrobial

substances stored in granules, to protect the host tissues from invading pathogens. Tear-film

neutrophils collected from the closed-eye environment have been shown to express high

levels of degranulation and activation leukocyte markers (such as CD66b and Mac-1). As

these cells may play a role in ocular inflammation and disease, it is important to determine

their functionality. However, large variations in response and collection numbers have been

observed previously. Limited knowledge also exists on the ability of tear film PMNs to

respond to cytokines and mount an oxidative response. Thus, the objectives of this thesis

were to develop a standard protocol to process tear-film PMNs to reliably conduct

functionality tests in vitro and to assess the production of reactive oxygen species (ROS) in

tear film PMNs. Specifically, 1) the sensitivity of tear-film PMNs to experimental

procedures (fixation, centrifugation and incubation) was investigated in terms of expression

of surface receptors via flow cytometry; 2) two different cell collection methods were

evaluated, and changes in the expression of surface receptors of tear-film PMNs when

exposed to interleukin-8 (IL-8) and phorbol 12-myristate 13-acetate (PMA) were examined

through flow cytometry; and 3) the ability of tear-film PMNs to generate ROS was assessed

iv

using luminol-enhanced chemiluminescence. The response of tear-film PMNs was also

compared to blood-isolated PMNs.

Up to 20 participants were recruited in this research to perform cell collection and

donate a small blood sample. A gentle eye wash method was used to collect cells from the

closed-eye environment, whereby participants washed their eyes with sterile phosphate

buffer saline (PBS) upon awaking at home, and collected the runoff into a sterile

polypropylene tube, which was then delivered to the lab within two hours. The patch-

OSCCA collection was also tested; participants slept at home and covered one of their eyes

with a patch. When they woke up in the morning, they came to the lab directly with one eye

covered, and cells were collected using the ocular surface cell collection apparatus (OSCCA),

which gently irrigates the ocular surface with warm PBS and collects the solution and cells

from a funnel into a centrifuge tube. Very few cells were obtained using the patch-OSCCA

collection method and thus this method was not pursued further in this research.

When assessing the effect of experimental procedures and measuring cell activation

upon stimulation with IL-8 and PMA (a PKC activator), changes in the expression of CD11b

(activation and adhesion leukocyte membrane receptor), CD16 (degranulation and

phagocytosis marker), CD66b (membrane receptor expressed upon cell degranulation), CD45

(leukocyte common antigen) and CD55 (complement activation marker) were characterized

by flow cytometry. ROS production in stimulated (PMA, lipopolysaccharides (LPS) or N-

Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear-film PMNs was

measured using luminol-enhanced chemiluminescence (CL). In all experiments, blood-

isolated PMNs were also used to allow for comparison in phenotype.

v

Fixation with paraformaldehyde (PFA) is an important step in flow cytometry.

Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (5-fold or

more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed

samples, while CD16 was the only receptor to undergo significant downregulation upon post-

staining fixation. Furthermore, it was found that an additional centrifugation step prior to

antibody incubation and long (4hr) incubation at 37oC significantly altered the expression of

membrane receptors with significant reduction in expression of CD11b, CD16 and CD55

when compared to control samples. Therefore, to preserve cell phenotype and cell integrity

of tear film PMNs, any additional centrifugation and incubation step should be avoided and

post fixation staining is recommended.

To gain a further understanding on the phenotype of tear-film PMNs, their ability to

respond to IL-8, a cytokine present in the tear film of the closed-eye environment, was

characterized via flow cytometry. The expression of surface receptors CD11b, CD16, CD55

and CD66b on tear-film PMNs remained relatively unchanged when exposed to IL-8,

whereas some changes in the level of expression of surface receptors were observed in

response to PMA but in a lower magnitude compared to blood-isolated PMNs.

The respiratory burst is one of the essential killing mechanisms for PMNs and is also

related to phagocytosis. PMA stimulation was able to induce ROS production (as measured

by chemiluminescence) in tear-film PMNs, and two distinct responder groups were observed,

where the high responder group produced significantly more ROS than the low responders.

LPS and fMLP failed to induce intracellular ROS production in tear-film PMNs although

fMLP-stimulated tear-film PMNs generated ROS extracellularly in the first three minutes.

vi

These results suggested that the signalling pathways downstream of PKC as well as the

NADPH oxidase were functional but that intracellular signalling pathways upstream the PKC

were impaired.

This thesis contributes new and important knowledge on tear film PMNs. We proved

that the gentle eye wash method is currently the most effective at home collection method.

In addition, our study demonstrated that experimental procedures can significantly affect the

expression of membrane receptor expression on tear-film PMNs, and if these processing

steps are not carefully considered, conclusions on the phenotype of tear-film PMNs can be

severely impacted. Our findings also suggest that the lack of response to stimuli may be due

to an impairment in the receptor-mediated intracellular signalling pathway and/or insufficient

substrates within the cell. Finally, our study on respiratory burst identified two type of

responders, which could have significant implications for microbial keratitis and contact

lens-induced infiltrates.

This thesis highlights the potential role of tear film PMNs in ocular health and

inflammation but more work is still needed to gain a better understanding of the phenotype of

tear film neutrophils in the closed eye environment and their underlying mechanisms of

activation.

vii

Acknowledgements

First of all, I would like to express my sincere appreciation to my supervisor, Dr.

Maud Gorbet, for giving me the opportunity to work on such a fantastic project. She gives

me invaluable leadership throughout this research. Her encouragement and patience make

me confident to communicate and present ideas. I appreciate for what she has offered.

My sincere thankfulness goes to my co-supervisor, Dr. Lyndon Jones, for his prompt

help and advice.

I would also like to thank my committee members, Dr. Brian Dixon and Dr. Paul

Murphy, for their support, help, and suggestions for this project.

Thank you to everyone who participated in my study. I would like to thank Doerte

Luensmann, who helped to perform numerous screening tests for the participants, and

Miriam Heynen, who drew blood for my experiments. I would like to acknowledge all the

grad students in optometry for their help on this project.

Furthermore, thank you to my lab members, Dana, Yasmin, Sadaf and Matthew, for

their help on lab practices and friendship. Thank you for giving me comfort when I was upset

and sharing the laugh with me. I am glad to work with you.

Lastly, I would like to thank my family for their support and love, especially my

grandmother. I would not be the person who I am today without her. Her love makes me feel

that I am the happiest girl in the world. She makes me stronger. Love you, grandma.

viii

To my beloved grandmother,

Xiangqin Wang.

ix

Table of Contents

AUTHOR'S DECLARATION ................................................................................................. ii

Abstract .................................................................................................................................... iii

Acknowledgements ................................................................................................................. vii

Table of Contents ..................................................................................................................... ix

List of Figures ........................................................................................................................ xiv

List of Tables ........................................................................................................................ xvii

List of Abbreviations ........................................................................................................... xviii

Chapter 1 Introduction .............................................................................................................. 1

Chapter 2 Neutrophil Population and Selected Activation Pathways ....................................... 7

2.1. Overview of neutrophils ................................................................................................. 7

2.2. Flow cytometry and CD markers used to phenotype neutrophils .................................. 8

2.3. Neutrophil Recruitment ................................................................................................ 12

2.4. Intracellular signaling pathways in neutrophils ........................................................... 15

2.5. Respiratory Burst in neutrophils .................................................................................. 18

2.6. Conclusion .................................................................................................................... 20

Chapter 3 The Heterogeneity of Neutrophils and Tear-film Neutrophils ............................... 22

3.1. Introduction .................................................................................................................. 22

3.2. Neutrophils in the lung ................................................................................................. 22

3.3. Neutrophils in the placenta ........................................................................................... 24

3.4. Neutrophils in the mouth .............................................................................................. 25

3.5. Neutrophils in the eye .................................................................................................. 27

x

3.5.1. Ocular Immune Privilege ....................................................................................... 27

3.5.2. Overview of the ocular environment ..................................................................... 28

3.5.3. Tear film and its components ................................................................................ 29

3.5.4. Tear-film neutrophils ............................................................................................. 32

3.5.5. The non-inflammatory phenotype of tear-film PMNs ........................................... 33

3.5.6. Tear-film PMNs and ocular surface diseases ........................................................ 35

3.6. Conclusion .................................................................................................................... 37

Chapter 4 Effects of Experimental Procedures on The Expression of Membrane Receptors on

Tear-film Neutrophils ............................................................................................................. 38

4.1. Introduction .................................................................................................................. 38

4.2. Materials and Methods ................................................................................................. 41

4.2.1. Reagents and Monoclonal Antibodies ................................................................... 41

4.2.2. Cell Collection ....................................................................................................... 41

4.2.3. Experimental procedure ......................................................................................... 42

4.2.4. Fixation .................................................................................................................. 43

4.2.5. Flow cytometry ...................................................................................................... 45

4.2.6. Statistics ................................................................................................................. 45

4.3. Results .......................................................................................................................... 46

4.3.1. The impact of fixation ........................................................................................... 46

4.3.2. The impact of incubation temperature ................................................................... 48

4.3.3. The impact of centrifugation ................................................................................. 50

4.4. Discussion .................................................................................................................... 52

xi

4.5. Conclusion .................................................................................................................... 57

Chapter 5 Comparison of Two Collection Methods and Investigation of the Response of

Tear-film Neutrophils to IL-8 ................................................................................................. 58

5.1. Introduction .................................................................................................................. 58


5.2.1. Reagents ................................................................................................................. 61

5.2.2. Study visits ............................................................................................................ 61

5.2.3. Gentle eye wash method ........................................................................................ 62

5.2.4. Patch-OSSCA Collection ...................................................................................... 62

5.2.5. Collection of blood-isolated PMNs ....................................................................... 64

5.2.6. Cell stimulation and staining with fluorescent antibodies ..................................... 65

5.2.7. Flow cytometry ...................................................................................................... 65

5.2.8. Statistics ................................................................................................................. 65

5.3. Results .......................................................................................................................... 66

5.3.1. Patch-OSSCA collection versus home collection ................................................. 66

5.3.2. Tear-film and blood-isolated PMNs responses to IL-8 and PMA in vitro ............ 68

5.3.3. Comparing the phenotype of tear-film PMNs between collection days for each

participant ........................................................................................................................ 74

5.4. Discussion .................................................................................................................... 78

5.5. Conclusion .................................................................................................................... 83

Chapter 6 Characterization of Respiratory Burst of Tear-film Neutrophils by Luminol-

Enhanced Chemiluminescence ............................................................................................... 85

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6.1. Introduction .................................................................................................................. 85


6.2.1. Materials ................................................................................................................ 88

6.2.2. Blood-isolated PMNs isolation: ............................................................................ 88

6.2.3. Tear-film PMNs collection .................................................................................... 89

6.2.4. Luminol-enhanced chemiluminescence ................................................................ 89

6.2.5. Statistics ................................................................................................................. 90

6.3. Results .......................................................................................................................... 91

6.3.1. ROS production in fMLP-stimulated tear-film and blood-isolated PMNs ............ 91

6.3.2. ROS production of LPS-stimulated tear-film and blood-isolated PMNs .............. 92

6.3.3. Production of ROS in PMA-stimulated tear-film PMNs and blood-isolated PMNs

......................................................................................................................................... 94

6.3.4. Comparison of area under the curve and endpoints between tear-film and blood-

isolated PMNs ................................................................................................................. 98

6.3.5. CL values at time zero in unstimulated samples. ................................................ 100

6.3.6. Participant with seasonal allergy ......................................................................... 101

6.4. Discussion .................................................................................................................. 102

6.5. Conclusions ................................................................................................................ 107

Chapter 7 Conclusions and Future Work .............................................................................. 109

7.1. Conclusions ................................................................................................................ 109

7.2. Future work ................................................................................................................ 111

7.2.1 Further characterization of tear-film PMNs phenotype – Granules in tear film

xiii

PMNs ............................................................................................................................. 112

7.2.2 Further characterization of tear-film PMNs phenotype – Apoptosis ................... 112

7.2.3 Further characterization of tear-film PMNs phenotype – Cytokine release ......... 113

7.2.4 Further characterization of tear-film PMNs phenotype – Inducing tear film PMNs

phenotype in blood-isolated PMNs with IL-8 exposure ................................................ 113

7.2.5 Further characterization of tear-film PMNs phenotype – Using an in vitro model to

characterize mechanisms involved in tear film PMNs phenotype ................................ 114

7.2.6 The role of tear-film PMNs in seasonal allergy ................................................... 115

7.2.7 The role of tear-film PMNs in lens-related ocular complications ........................ 116

7.2.8 The role of tear-film PMNs in lens-related ocular infections – in vivo clinical

studies ............................................................................................................................ 116

7.2.9 The role of tear-film PMNs in lens-related ocular infections – in vitro model .... 117

Letter of Copyright Permissions ........................................................................................... 118

Bibliography ......................................................................................................................... 121

Chapter 1 references .......................................................................................................... 121







xiv

List of Figures

Figure 1: Flow cytometry. ....................................................................................................... 10

Figure 2 - Overview of neutrophil recruitment cascade for PMNs migrating from the blood

vessels to the peripheral tissue. ............................................................................................... 14

Figure 3 – Intracellular signaling pathways in response to stimulation of PMNs with fMLP

and PMA. ................................................................................................................................ 16

Figure 4 - The overall formation of ROS by a phagocytic cell. ............................................. 20

Figure 5 - Anatomy of the human eye. ................................................................................... 29

Figure 6 – a) An old view of three layered tear-film and b) the polar and non-polar phases

within the lipid layer ............................................................................................................... 30

Figure 7 - The modern view of the tear film ........................................................................... 31

Figure 8 - Illustration of the at-home tear-film PMNs collection process. ............................. 42

Figure 9 - Experimental flow chart. ........................................................................................ 45

Figure 10 - Effect of PFA on the expression of cell activation surface markers on tear-film

PMNs. ..................................................................................................................................... 47

Figure 11 – Effect of exposure to 37oC for 4h on the expression of cell membrane activation

markers on tear-film PMNs. ................................................................................................... 49

Figure 12 – Effect of centrifugation on the expression of cell membrane activation markers

on tear-film PMNs. ................................................................................................................. 51

Figure 13 - Schematic diagram of the study visits. ................................................................. 62

Figure 14 –OSCCA setup and the eye patch.. ........................................................................ 64

Figure 15 - The total cells collected using the home collection method with the gentle eye

xv

wash (EW) and the patch-OSCCA collection method (PC). .................................................. 66

Figure 16 - Schematic diagram of the revised study visits ..................................................... 67

Figure 17 - Total cell numbers collected using the gentle eye wash method over a one-month

period. ..................................................................................................................................... 68

Figure 18 - The expression of surface receptors of tear-film PMNs after sleep following

stimulation with a) IL-8 and b) PMA. .................................................................................... 69

Figure 19 - The expression of surface receptors of blood-isolated PMNs following

stimulation with a) IL-8 and b) PMA. .................................................................................... 70

Figure 20 - The expressions of surface receptors of blood-isolated PMNs and tear-film PMNs

following stimulation with IL-8. ............................................................................................. 71

Figure 21 - The expression of surface receptors of blood-isolated PMNs and tear-film PMNs

following stimulation with PMA. ........................................................................................... 72

Figure 22 – Change in IL-8 and PMA stimulation of tear-film PMNs collected at the

beginning and end of the month. ............................................................................................. 75

Figure 23 – Change in the level of unstimulated expression of membrane receptors of tear-

film PMNs collected at the beginning and end of the month. ................................................ 77

Figure 24 – Examples of chemiluminescence kinetic response recorded with tear film PMNs

when exposed to a) PMA, b) fMLP and c) LPS. .................................................................... 90

Figure 25 - Changes in chemiluminescence of tear-film and blood-isolated PMNs when

exposed to fMLP. .................................................................................................................... 92

Figure 26 - Changes in chemiluminescence of tear-film and blood-isolated PMNs when

exposed to LPS. ...................................................................................................................... 93

xvi

Figure 27 – Changes in chemiluminescence of tear-film and blood-isolated PMNs when

exposed to PMA. . ................................................................................................................... 95

Figure 28 - Changes in CL of PMA-stimulated tear-film PMNs of high responders (HR) and

low responders (LR). .............................................................................................................. 98

Figure 29 – Comparison of area under the curve (AUC) and the ratio of changes at the

endpoint (t = 120 min) between tear-film and blood-isolated PMNs for all three stimuli ... 100

Figure 30 – Kinetic changes in CL of tear-film PMNs collected from the allergy participant

over 60 min compared to asymptomatic/healthy participants .............................................. 102

Figure 31 – An in vitro culture model to mimic the extravasation on blood-isolated PMNs.

............................................................................................................................................... 115

xvii

List of Tables

Table 1 - CD markers commonly used to characterize the phenotype of PMNs. ................... 12

Table 2 – Components of the artificial tear solution ............................................................... 43

Table 3 – Summary of changes in the expression of surface receptors on tear-film PMNs

(processed versus unprocessed sample). ................................................................................. 52

Table 4 - Change in level of expression of receptors on tear film neutrophils following

fixation. ................................................................................................................................... 53

Table 5 - Expression of selected membrane receptor on unstimulated tear-film and blood-

isolated PMNs. ........................................................................................................................ 73

Table 6 – Components of HBSS 13. ........................................................................................ 90

Table 7 - Summary table of ROS generation potentials for high responders and low

responders. .............................................................................................................................. 97

Table 8 - ROS production in unstimulated tear film and blood-isolated PMNs ................... 101

xviii

List of Abbreviations

AA: Arachidonic acids

ARDS: Acute respiratory distress syndrome

AUC: Area under the curve

BAL: Bronchoalveolar lavage

CD: Cluster of differentiation

COPD: Chronic obstructive pulmonary disease

CP: Chronic periodontitis

DAG: Diacylglycerol

DAF: Decay-accelerating factor

DED: Dry eye disease

DMEM: Dulbecco’s modified eagle media

DNA: Deoxyribonucleic acid

EW: Gentle eye wash method

fMLP: N-Formyl-L-methionyl-L-leucyl-L-phenylalanine

G-CSF: Granulocyte colony-stimulating factor

GM-CSF: Granulocyte-macrophage colony-stimulating factor

GPCR: G-protein-coupled receptor

GPI: Glycosylphosphatidylinositol

CR3: Complement receptor

HBSS: Hanks's balanced salt solution

xix

hCECs: Human corneal epithelial cells

HLDA workshop: Human leukocyte differentiation antigens workshop

HO-: Hydroxyl radical

HOCl: Hypochlorous acid

HR: High responders

!"#": Hydrogen peroxide

IL-3: Interleukin-3

IL-8: Interleukin-8

IP3: Inositol triphosphate

KM: Keratinocyte serum-free medium

LDGs: Low-density granulocytes

LPS: Lipopolysaccharides

LR: Low responders

Mac-1: Macrophage-1 antigen

MAPK: Mitogen-activated-protein kinase

MEK1: Mitogen-activated protein kinase kinase 1

MEKK-1: Mitogen-activated-protein kinase kinase kinase 1

MFI: Mean fluorescent intensities

MGD: Meibomian gland dysfunction

MK: Microbial keratitis

MPO: Myeloperoxidase

NADPH oxidase: Nicotinamide adenine dinucleotide phosphate oxidase

xx

NDGs: Normal-density granulocytes

NETs: Neutrophil extracellular traps

O2- : Superoxide anion

OA: Ocular allergy

OSCCA: Ocular surface cell collection apparatus

PA: Phosphatidic acid

PAC: Perennial allergic conjunctivitis

PBS: Phosphate-buffered saline

PC: Patch-OSCCA collection method

PFA: Paraformaldehyde

PKC: Protein kinase C

PLA2: Phospholipase A2

PLC: Phospholipase C

PLD: Phospholipase D

PI3K: Phosphatidylinositol 3-kinase

PMA: Phorbol-12-myristate-13-acetate

PMN: Polymorphonuclear neutrophils

RNA: Ribonucleic acid

ROS: Reactive oxygen species

rTEM: Transendothelial cell migration

SAC: Seasonal allergic conjunctivitis

SD: Standard deviation

xxi

sIgA: Secretory immunoglobulin A

ST: Screening test

STMR: Seven transmembrane-spanning receptor

TNF-a: Tumor necrosis factor a

1

Chapter 1

Introduction

The anterior segment of the eye represents a unique physiological environment due to

its specific characteristics linked to immune privilege1,2. Inflammatory cells are believed to

be highly regulated because of the required balance between protection of the eye against

pathogens and preservation of the integrity of the corneal epithelium1,2. In the context of an

immune privileged-site, immune and inflammatory cells (lymphocytes, neutrophils,

monocytes, etc.) are only to be introduced to the local environment when an infection or a

wound is present. However, in the anterior eye, a significant influx of neutrophils, combined

with an increase in complement activity, metalloproteinases and cytokines, have been

detected in the closed-eye environment (during sleep) even in the absence of any wound or

infection1,3. This neutrophil population is often referred to as “tear-film neutrophils” to

reflect the local environment where their presence has been clearly identified4. Tear-film

neutrophils were first discovered in 1989 by Wilson et al., where they observed that the

number of leukocytes collected after sleep in the morning was significantly higher than in the

afternoon3. The limited number of cells collected prevented assessing cell functionality.

Thus, researchers, at that time, focused on comparing the numbers of tear-film neutrophils

recovered between open-eye and closed-eye tears3, and also between tears collected from

lens-wearers and non-lens wearers, to determine if the presence of contact lenses altered the

recruitment of tear-film neutrophils. The increase in cell numbers, chemokines and

inflammatory mediators in the closed-eye environment have led researchers to believe that

tear-film neutrophils are present to eliminate pathogens that may be “trapped” during closed

2

eye5,6. More efficient methods for ocular cell collection and characterization have renewed

the interest on the role of tear-film neutrophils in ocular health.

Non-invasive methods used to collect cells from the ocular surface have evolved over

the years and have been used to harvest not only neutrophils but also human corneal

epithelial cells (hCECs). The “non-contact corneal irrigation chamber” and disposable glass

micro-capillary tubes were used in 19867 and 19988, respectively, to collect cells, but less

than 200 hCECs and 8000 leukocytes were usually obtained, preventing any cell

phenotyping. The ocular surface cell collection apparatus (OSCCA), developed by Peterson

et al. allowed researchers to harvest more cells from the ocular surface compared to previous

methods9. While effective in cell collection, as it can collect over hundreds of thousands of

neutrophils from the ocular surface10, the cost involved with overnight studies limited the use

of the OSCCA for collection of tear-film neutrophils, as participants were required to sleep at

the clinic. A new method, using a gentle eye wash, was thus designed by Gorbet et al., and

allowed participants to collect tear-film neutrophils at home1.

Collecting more cells from the closed-eye environment has enabled researchers to

start characterizing the phenotype of tear-film neutrophils. The study by Gorbet et al

indicated that tear-film neutrophils may have a non-inflammatory phenotype, as they were

unable to respond to inflammatory stimuli through upregulation of membrane receptors

commonly associated with activation1. Furthermore, Gorbet and Willcox showed that tear

film neutrophils have an impaired ability to kill bacteria11. These findings suggest that the

function of tear film neutrophils upon collection from the eyes after sleep may be diminished.

This impaired function of tear-film neutrophils may be due to the presence of tear proteins,

such as lactoferrin, or the hypoxic condition of the closed-eye environment. However,

3

efforts to mimic the conditions of the closed-eye environment failed to induce the same non-

inflammatory phenotype in blood-isolated neutrophils, and further suggested the

heterogeneity of neutrophil populations4.

Tear-film neutrophils have been shown to express high level of degranulation and

activation leukocyte markers (CD66b and Mac-1, respectively, which will be further

explained in chapter 2) but low level of L-selectin1,12. Similar observations have been made

on the phenotype of neutrophils that have migrated from the blood into tissues, such as the

healthy lungs13, the mouth14 and the placenta15. Recent observations of tear-film neutrophils

collected after various times of exposure to the closed-eye environment have also led

Postnikoff et al to hypothesize that these cells have been already activated in their early

recruitment to the ocular surface12. These recent studies on tear film-neutrophils thus tend to

suggest a complex phenotype - with a yet undefined origin.

To characterize inflammatory phenotype of leukocytes, cells are activated in vitro.

The stimuli used to characterize the response of tear-film neutrophils have been phorbol-12-

myristate-13-acetate (PMA; a potent synthetic chemical activator of protein kinase C),

lipopolysaccharides (LPS; a bacterial endotoxin), N-Formyl-L-methionyl-L-leucyl-L-

phenylalanine (fMLP; a chemotactic peptide), and calcium ionophore1,12,16. However, these

are not all physiological stimuli nor representative of other stimuli that tear-film neutrophils

may encounter in the ocular environment. Cytokines, such as tumor necrosis factor a (TNF-

a)17, granulocyte-macrophage colony-stimulating factor (GM-CSF)18, and interleukin-8 (IL-

8)19 have been recognized to activate blood-isolated neutrophils, however little is known on

the response of tear-film neutrophils to these or other cytokines. Among these more

physiological stimuli, IL-8 is a chemokine for neutrophils, leading to activation and

4

migration of blood leukocytes to infected sites through the actions of L-selectin and Mac-

120,21, and high levels of IL-8 have been observed in the closed-eye environment8.

Characterizing the response of tear-film neutrophils to IL-8 would provide information on

their activation state in the closed-eye environment as well as contribute further evidence

towards their phenotypic difference from blood leukocytes.

The respiratory burst, via the release of reactive oxygen species (ROS), is also part of

the leukocyte inflammatory response and is one of the efficient ways for neutrophils to kill

pathogens. The mechanisms that blood-isolated neutrophils used to generate ROS have been

well characterized, and it is known that blood-isolated neutrophils used different intracellular

signaling pathways to respond to various stimuli22–24. While the ocular surface is exposed to

various pathogens, there is currently limited knowledge on ROS generation with tear-film

neutrophils. Hume et al. have used blood-isolated neutrophils to characterize ROS

generation upon exposure to ocular isolated bacteria, Serratia marcescens, to study the

potential underlying mechanisms of microbial keratitis25. However, since recent research has

indicated that blood-isolated and tear- film neutrophils exhibit different phenotypes, it may

not be sensible to use blood-isolated neutrophils to assess mechanisms of inflammation from

the ocular surface. Furthermore, using flow cytometry, the ability of tear-film neutrophils to

generate ROS was reported to be significantly lower than blood-isolated neutrophils16.

However, flow cytometry provides limited insights into the process and potential

mechanisms of ROS generation by tear-film neutrophils. The extent and kinetics of ROS

generation can be characterized by luminol-enhanced chemiluminescence and could

contribute significant knowledge to the mechanisms of ROS generation in tear-film

neutrophils and allow us to gain a better understanding of ocular inflammation.

5

As can be seen from the number of recent studies, tear-film neutrophils (both from

the closed- and open-eye environment) are emerging as a new source of knowledge on

inflammation at the ocular surface1,12,16,26,27. In addition, tear-film neutrophils may play a

role in some ocular diseases, such as dry eye disease12,28 and seasonal ocular allergy29, but

their interactions with other immune cells and their activation mechanisms need to be further

examined. Different protocols have been used post-collection of neutrophils from the ocular

surface, which may lead to variations in results and affect characterization of cell phenotype.

Various collection protocols have also been used previously and no systemic study has yet

been undertaken to investigate the sensitivity of tear-film neutrophils to experimental

procedures1,8,12,30,31. This significantly affects our ability to reliably use these cells for the

development of diagnostic or screening tools or as a potential source of cells for in vitro

experimentation. Centrifugation, used to concentrate cells and to wash out any unwanted

substances, and fixation, used to crosslink the fluorescent-conjugated antibodies to their

membrane receptors, are usually involved in immunophenotyping experiments. The impact

of preparation procedures on blood cells have been extensively investigated, but, based on

our knowledge, this has not yet been studied with tear-film neutrophils. There is thus a need

to investigate how cell collection and processing may affect cell phenotype and to develop

standardized protocols that can be used to reliably collect and phenotype tear-film

neutrophils.

The overarching hypothesis of this thesis is that tear-film neutrophils are sensitive to

experimental procedures and exhibit a unique phenotype different from their counterpart

blood leukocytes. The objectives of this thesis from the experimental procedure perspective

were thus to compare two collection methods, using OSSCA and gentle eye wash, to

6

determine which could yield most cells and to assess the impact of centrifugation and

fixation on the expression of membrane receptors on the collected tear-film neutrophils.

From the perspective of further characterizing the phenotype of tear-film neutrophils, the

objectives were to assess their response to cytokine IL-8 as well as their ability to generate

ROS under various stimuli.

This thesis begins with an introduction on neutrophils, such as transmigration process

and mechanisms for respiratory burst, in chapter 2, and provides more information on the

heterogeneity of neutrophils, the ocular environment, and tear-film neutrophils in chapter 3.

Chapter 4 reports on the sensitivity of tear-film neutrophils to experimental procedures

involving centrifugation and fixation steps. Chapter 5 compares the two collection methods

and presents the response of tear-film neutrophils to IL-8. Chapter 6 compares the ability of

tear-film neutrophils and blood-isolated neutrophils in ROS generation upon stimulation

using the luminol-enhanced chemiluminescence assay. Chapter 7 presents conclusions and

recommendations for future work.

7

Chapter 2

Neutrophil Population and Selected Activation Pathways

2.1. Overview of neutrophils

Innate immunity is an older evolutionary mechanism protecting the host tissue against

foreign particles, and involves many inflammatory cells, such as, granulocytes (also known

as polymorphonuclear leukocytes), mast cells, and macrophages1. Neutrophils

(polymorphonuclear neutrophils, PMNs) are one of the populations of granulocytes and are

the first cells to arrive at infected sites2. The development of mature PMNs from

hematopoietic stem cells in the bone marrow is a complex process involving multiple steps2.

The entire process is highly regulated by the cooperation of various transcription factors,

PU.1, and C/EBP$3,4, and also hematopoietic growth factors, granulocyte colony-stimulating

factor (G-CSF), interleukin-3 (IL-3), and granulocyte-macrophage colony factor (GM-CSF)2.

In healthy individuals, there are approximately 1011 PMNs circulating in blood everyday2. A

large number of mature PMNs can be found not only in the bone marrow, but also in the

liver, spleen, and lungs, and these are referred to as marginated intravascular granulocyte

pools5. However, the size of the pulmonary granulocyte pool is controversial, because blood

needs to transit through the lungs to be oxygenated and thus margination can be difficult to

assess; concentrated PMNs can be found in the healthy lungs6 but in a small number5.

Unlike other leukocytes population, such as macrophages and lymphoid cells7, the

neutrophil population, which represents approximately 60% of all white blood cells in

bodies, is believed to be homologous, which means they do not hold a diversity in

differentiation and activation, due to their short lifespan, their inability to transmigrate back

to blood and their reduced transcriptional activity8–10. However, a recent experiment by

8

Pillay et al., in which they traced PMNs in blood and bone11,12, showed that PMNs can

circulate in the blood for up to 5.4 days12. This challenged the traditional view of their

lifespan, which is thought to be approximately 8 hours in humans2,5. In addition to this,

using an in vitro flow model, Buckley et al. explored the fact that PMNs may be able to re-

transmigrate back to blood13. These controversies around neutrophils draw our attention to

their potential for heterogeneity, which needs to be further examined. Since many review

papers exist on the phenotypic difference between PMNs in people affected by various

diseases versus healthy individuals, we aimed to focus more on the heterogeneous neutrophil

populations that can be found in different parts of the body, such as the lungs, the placenta,

the mouth, and the eye. The information provided in this chapter provides some valuable

background, as we aimed to characterize the phenotypes of tear-film PMNs and investigate

their capability to induce the respiratory burst when exposed to different stimuli.

2.2. Flow cytometry and CD markers used to phenotype neutrophils

The cluster of differentiation (CD) is used to identify, discover, and organize different

cell surface molecules present mainly in leukocytes14. It was developed by scientists in the

human leukocyte differentiation antigens (HLDA) workshop held in Paris in 198514. Apart

from identifying or coding the cell surface molecules, the HLDA workshop also provided a

forum for discussing the function and distribution of molecules and epitopes15. CD markers

are utilized to identify and characterize leukocytes through the binding to their corresponding

cell surface molecules, which act as receptors or ligands.

The phenotype of PMNs, describing the expression of receptors on their cell

membranes, can be evaluated using (1) the immunophenotyping method16 that is quantitative

and can measure differences between samples, or (2) the immunocytochemistry method17

9

which allows visualizing targeted membrane receptors on the cell.

Flow cytometry (immunophenotyping) is the common method used to measure

multiple characteristics of individual cells, including cell size, granularity or cytoplasmic

complexity, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and membrane-bound

and intracellular proteins18. It was largely used in this research to identify and assess PMNs

activation, since when PMNs are stimulated, membrane receptors can either be upregulated

or downregulated. Antibodies (or CD markers) conjugated with fluorescent molecules bind to

their corresponding antigen and in this thesis, we focussed on selected membrane receptors

of cell activation.

Acquisition of data through flow cytometry is depicted in Figure 1. Antibody-stained

cells are aspirated into the flow cytometry microfluidics system and aligned into a single file

line through laminar flow (step 1). Once in the flow cell, the cells are hit by a laser beam

(step 2), the light will scatter based on the size and granularity of the cells (step 3) and the

light will also excite any fluorescent molecules on cell membranes or inside the cell (step 4).

Optic detectors will capture the light information (side scatter, forward scatter and

fluorescent intensities) and this will be processed by the various electronic components and

translated into quantitative values (step 5).

10

Figure 1: Flow cytometry. (1) sample intake with fluorescently labelled cells, (2) flow cell where laser illuminates cells one at a time, (3) detectors for forward and side scatter, (4) detectors for fluorescence, (5) data processing. Adapted from ThermoFisher/Invitrogen, www.thermofisher.ca

As neutrophils have a range of receptors for identification and activation, several

markers need to be used to characterize neutrophil populations. It is well established that

PMNs express CD11b, CD15, CD16, CD62L, and CD66b on their membranes19–22. CD

markers commonly used to identify and characterize the phenotype of PMNs are summarized

and listed in table 1.

In this thesis, we specifically examined the expressions of CD11b, CD16, CD45,

CD55, and CD66b on tear-film PMNs. CD11b is an activation and adhesion marker and

combines with CD18 to form the macrophage-1 antigen (Mac-1) or complement receptor

(CR3), which is a member of the b2 family of integrins23. CD11b plays a crucial role in

PMNs transmigration from the blood vessel to the site of infection and also in mediating cell

immune responses24. The upregulation of CD11b occurs early in the cell activation process

and is used to determine the activation state of leukocytes2. CD16, a member of the Fc

11

gamma receptors for immunoglobulin, has been shown to be associated with the

phagocytosis and degranulation of PMNs25. During PMNs activation, CD16 is shed off

during neutrophil elastase release, and this reduction in CD16 expression has also been

shown to be correlated with cell apoptosis26,20. CD55 is a glycosylphosphatidylinositol

(GPI)-anchored decay-accelerating factor (DAF) and is one of the regulators of complement

activation27. CD55 is mainly stored in secretory vesicles and has a low expression on resting

PMNs in the circulation. It has been shown to be upregulated after activation and can help to

differentiate between bacterial and viral infection27. CD66b is a degranulation marker, and

its expression increases when cells are activated23. CD45 is a common leukocyte maker and

is mainly used to identify leukocyte populations.

12

Table 1 - CD markers commonly used to characterize the phenotype of PMNs. CD marker – Antigen Leukocyte function assessed

CD11b (Mac-1)11,19–22,28–31 Adhesive cell interaction, a marker for neutrophil

activation

CD1419,20,32 Binding receptor for LPS, a marker for phagocytosis

CD1519,20,33 Mature neutrophils marker

CD1620–22,26,30,34–37 Fc gamma type III receptor, a marker for degranulation

and phagocytosis

CD17021,30 Immunoregulation marker

CD1778,9,30 Marker for regulating transmigration across the

endothelium (adhesion marker)

CD18121,22,34 Receptor for type I IL-8

CD1829,21,22,34 Receptor for type II IL-8

CD3221,22,26,36,38 Fc gamma type II receptor, a marker for phagocytosis

CD4339–42 A marker for PMNs reactive oxygen species production

and phagocytosis

CD4511,19–22,28–31 Common leukocyte marker

CD54 (ICAM-1)13,22 Adhesion marker

CD5530 Complement regulation marker

CD62L19,20,22,38,43,44 L-selectin

CD6330,33 Marker for azurophil granules

CD66b11,19–22,28–31 Degranulation marker

CD8821,22 Receptor for C5a ligand

2.3. Neutrophil recruitment

Recruitment of PMNs from blood to the peripheral tissue is caused by the presence of

inflammatory mediators, such as histamine, chemokines, cytokines, and lipid mediators11,31.

The process known as extravasation usually consists of multiple steps for PMNs migrating

across the blood vessel walls, including tethering (capturing), rolling, adhesion, crawling, and

13

eventually transmigration (Figure 2)11,31. Integrins and selectins are the key molecules

involved in these steps11,31,43,45. Upon stimulation by pathogens, endothelial cells lining the

blood vessels upregulate the expression of selectins on their luminal surface45. Free-flowing

PMNs can then be captured on the endothelium through the interaction between the

leukocyte glycoproteins and their corresponding selectins (P- or E-selectin) on the

endothelium43,46. Then, attached PMNs roll along with the blood vessel through the

interactions between E-selectins and leukocyte glycoproteins11,46. This rolling step is not

sufficient for PMNs to firmly attach to endothelial cells due to the shear force from blood

flow46. The binding of the integrin Mac-1 (also known as CD11b) expressed by PMNs and

immunoglobulin superfamily members (ICAM-1) expressed by endothelial cells help to

mediate leukocyte arrest43. L-selectin (CD62L) expressed by PMNs play an essential role in

secondary tethering, helping to bind free-flowing neutrophils to already adherent PMNs47.

Next, PMNs roll along the blood vessel while maintaining adhesion to the endothelium, new

bonds to the endothelium are formed as soon as the existing bonds are released during the

crawling process11. This crawling step is dependent on ICAM-1 and Mac-148. PMNs

migrate to local tissue either through junctions between adjacent endothelial cells

(paracellular) or through the body of the endothelial cell (transcellular), but paracellular

transmigration is predominant11,43,45. Transmigration is associated with increased levels of

intracellular Ca2+ and is supported by the binding of PMNs integrins and endothelium

expressed ligands (ICAM-1 and VCAM-1) via re-arranging or reducing junctional molecules

(such as VE-cadherin)11,43,45, resulting in fewer barriers, which makes it easier for PMNs to

migrate into the tissue.

The extravasation process significantly alters the PMNs expression of certain

14

membrane receptors. There is a drastic reduction in the expression of L-selectin (CD62L) as

it is being shed during the interaction with the endothelial cells, but extravasated PMNs have

a significant increase in %1 and %2 integrins (Mac-1) and recognition receptors (CXCR4)49.

After transmigration, PMNs are programmed to undergo apoptosis after they execute their

inflammatory functions in the tissue, and their fragments are phagocytosed by macrophages9.

However, it has been shown that some PMNs can transmigrate from the peripheral tissue

back to the blood vessels in zebrafish and mice, in a phenomenon called reverse

transendothelial cell migration (rTEM)13. It is interesting to note that these rTEM PMNs now

circulating back in blood exhibit a different phenotype from the normal circulating blood

PMNs. rTEM PMNs have significantly higher expression in Mac-1 (%2 integrins) but lower

expression of L-selectin13.

Figure 2 - Overview of neutrophil recruitment cascade for PMNs migrating from the blood vessels to the peripheral tissue 11. Each step is mediated by the interaction between PMNs (integrins and leukocyte glycoprotein) and endothelial cells (selectins). PMNs transmigrate to local sites either via the paracellular way (through junctions between the adjacent endothelial cells), or via the transcellular way (through the body of the endothelial cells) which is not shown in this figure. Reprinted with permission from: Kolaczkowska, E. & Kubes, P. Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 13, 159–175 (2013).

15

2.4. Intracellular signaling pathways in neutrophils

PMNs execute their functions mainly through the binding of the stimulus to its

corresponding membrane receptor/ligand. Transmembrane signaling is crucial to control the

intracellular events which direct the PMN functional responses. There is a wide range of

stimuli that activate PMNs, such as cytokines (in this research, IL-8 was used), chemotactic

stimuli (such as N-formyl-met-leu-phe (fMLP), used in this research), chemical stimuli (such

as Phorbol 12-myristate 13-acetate (PMA), used herein), bacterial endotoxins (also known as

lipopoly-saccharides (LPS)). In this section, the intracellular signaling pathways activated in

response to stimulation of PMNs with fMLP and PMA are mainly discussed and are depicted

in the schematic diagram in Figure 3.

16

Figure 3 – Intracellular signaling pathways in response to stimulation of PMNs with fMLP and PMA. After fMLP binds to its receptor (G-coupled protein receptor), multiple pathways are activated. The black square represents the components that can be activated by PMA via the direct binding. STMR: seven transmembrane-spanning receptor; PLC: phospholipase C; DAG: diacylglycerol; IP3: Inositol triphosphate; PKC: protein kinase C; MEKK-1: mitogen-activated-protein kinase kinase kinase 1; MEK1: mitogen-activated protein kinase kinase 1; MAPK: mitogen-activated-protein kinase; PLA2: phospholipase A2; AA; arachidonic acids; PLD: phospholipase D; PA: phosphatidic acid; PI3K: phosphatidylinositol 3-kinase.

fMLP receptor is a seven transmembrane-spanning and pertussis-toxin sensitive G-

protein-coupled receptor (GPCR)50. Extensive studies show that the interaction between

fMLP and the GPCR activates phospholipase C (PLC) and phospholipase D (PLD)2,50–53.

Phospholipase A2 (PLA2) is activated by coupling to the receptor via a G protein, Gp54. The

activation of PLD is mediated by the ADP-ribosylation factor and Rho proteins which are

stimulated by G proteins55. PLC hydrolyzes phosphoinositide into two second messengers,

diacylglycerol (DAG) and Inositol triphosphate (IP3)2,50. DAG is a protein kinase C (PKC)

17

activator, whereas IP3 regulates the mobilization of extra- and intracellular calcium2,52. PLD

uses phosphatidylcholine as the substrate to generate the lipid messenger phosphatidic acid

which can be further converted to DAG2,56, which can activate PKC later on. There are two

types of PKC which are conventional PKC requiring both DAG and Ca2+ to be activated, and

nPKC only requires DAG56. Activated PKC has a variety of functions, for example,

initiating the respiratory burst (see below for details) via the phosphorylation of the

nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) and upregulation of

adhesion receptors such as Mac-1 (CD11b). In addition to the pathway involving PKC,

several studies show that fMLP is able to stimulate Ras and Raf which eventually leads to the

activation of p42/44 (ERK) mitogen activated protein kinase (MAPk)50–52. Activated MAPk

phosphorylates phospholipase A2 (PLA2)52, causing the release of arachidonic acids (AA), is

correlated with the generation of superoxide anion2. The general outcome of this MAPk

pathway is to increase transcription through phosphorylation of transcription factors.

Furthermore, fMLP activates p38 kinase which is one of the mitogen protein kinases51. This

kinase cascade plays an essential role in altering PMNs responses, such as regulation of gene

expressions57, increased synthesis and release of cytokines, and release of superoxide anion51.

Tyrosine kinase 2 and phosphatidylinositol 3-kinase (PI 3-kinase)56 are the two other kinases

that can be activated by fMLP to trigger cellular events, such as increase in chemotaxis.

The intracellular mechanisms for PMA is quite direct as PMA enters the cell directly

and binds to PKC, which then upregulate CD11b/CD18 complex58 and activate NADPH

oxidase to induce the respiratory burst59. PMA is also able to phosphorylate and activate

both p38 and p42/44 kinases51. Karlsson et al. showed that the intracellular oxidase response

to PMA is dependent on PI 3-kinase which challenged the previous findings that PMA-

18

induced superoxide release is PI 3-kinase independent59.

2.5. Respiratory Burst in neutrophils

Phagocytic cells, such as monocytes/ macrophages and PMNs, use the oxygen-

dependent killing mechanism referred to as the respiratory burst to protect host tissues from

invasion of foreign pathogens60,61. Molecules that are synthesized in this process and are

involved in the oxygen-dependent killing mechanism are referred to as reactive oxygen

species (ROS) and include superoxide anions (O2- ), hypochlorous acid (HOCl) and hydroxyl

radicals (HO-). During phagocytosis, a significant increase in oxygen uptake in neutrophils

and monocytes was observed by Sbarra and Karnovsky62. This increased consumption of

oxygen is not due to respiration, but is related to an oxidase-reducing molecular oxygen to

the superoxide anion, the primary product of respiratory burst62, 2. They also noted a positive

correlation between oxygen consumption and concentration of particles available for

engulfing/phagocytosis62. The ability of PMNs to generate ROS is not equivalent to their

capability for phagocytosis2. Patients with chronic granulomatous disease can phagocytose

microbes but cannot produce ROS63. Since some of the ROS are strong anti-microbial

molecules, it may also cause damage to host tissues which can lead to many inflammatory

diseases, for example, the contagious bovine pleuropneumonia (CBPP)61,64. Excessive

production of ROS is not only harmful for tissues but also detrimental for cells, as it can

result in T lymphocytes hyporesponsiveness65.

Several enzymes cooperate together to generate the end products of the respiratory

burst. First, an oxidase reduces molecular oxygen (O2) to the superoxide anion (O2- ) which

initiates the respiratory burst66. Since NADPH is used as the electron donor in this reaction,

the enzyme is known as NADPH oxidase. NADPH oxidase consists of a membrane bound

19

component which is the b-type cytochrome and four cytosolic proteins61. Cytosolic proteins

contain the NADPH acting as the electron donor. The b-type cytochrome is embedded

within both the plasma membranes (5%) and the membranes of intracellular granules and

vesicles (95%)67. Once neutrophils are activated, cytosolic proteins translocate to the b-type

cytochrome. Then, electrons are transferred from NADPH, in the cytosolic proteins, over the

membranes and delivered to the molecular oxygen present sites, the phagosomes or the

extracellular milieu, by b-type cytochrome68. NADPH oxidase catalyses the following

reaction (equation 1) in which superoxide anion is the predominant product61.

NADPH + H, + 2O" → NADPH, + 2H, + 2O"0 (1)

Then, superoxide spontaneously dismutates to form hydrogen peroxide (equation 2),

where superoxide dismutase catalyzes the reaction2.

2O"0 + 2H, → O" + H"O" (2)

Other more reactive ROS are further generated via the actions of nitric oxide synthase

and myeloperoxidase (MPO)61. The overall generation of ROS is depicted in Figure 4.

20

Figure 4 - The overall formation of ROS by a phagocytic cell 69. Superoxide (120) and hydrogen peroxide (4212)are less reactive, while hypochlorous acid (HOCl) and OH radical are more reactive. © 2017 Nguyen, Green and Mecsas.

2.6. Conclusion

Following maturation in the bone marrow, PMNs are released into the circulation

where they are distributed into two pools, the circulation pool and the marginated pool2.

PMNs are short-lived cells, but their actual life-span in blood is currently controversial. The

neutrophil population is heterogeneous, meaning PMNs exhibit distinct phenotypes under

both pathological and normal physiological conditions. Immunopheynotyping via flow

cytometry is the common method researchers often used to characterize PMNs phenotypes

through the expression of surface receptors or antigens. Different intracellular signaling

pathways, which involves multiple phospholipases and kinases, are used by PMNs upon

exposure to stimuli in order to execute their cell functional responses, for example, the

21

respiratory burst. The hallmark of the respiratory bust is the substantial increase in the

consumption of oxygen which is used to generate ROS. Several key enzymes have to work

cooperatively to produce the strong antimicrobial agents.

22

Chapter 3

The Heterogeneity of Neutrophils and Tear-film Neutrophils

3.1. Introduction

Distinct phenotypes of PMNs have been detected in various pathological conditions,

as well as in different tissues of healthy individuals. Thus, neutrophils don’t exist as one type

of cells but are now considered as heterogeneous neutrophil populations. Their heterogeneity

in pathological conditions will be briefly illustrated by using sepsis as an example. In severe

systemic inflammation induced by sepsis, the leading cause of death is the demonstration of

immune suppression in patients, which makes them more susceptible to infections, despite

the massive presence of circulating PMNs in blood1. This may suggest that the immune

suppression observed in sepsis patients is due to an altered function in PMNs, where they

exhibit an inability to kill pathogens, inhibit other immune cells (T cells)2, and eventually

suppress the immune response. In this chapter, we will focus mainly on the phenotypes of

PMNs found in different tissues, the lung, the placenta, the mouth and the eye.

3.2. Neutrophils in the lung

PMNs can be detected in the lung alveoli under both physiological and pathological

conditions3,4. Resting blood PMNs have a limited ability to respond to stimuli. The process,

in which PMNs change to a more active state, is referred to as ‘priming’, whereby PMNs are

exposed to low concentration of an inflammatory molecule5, allowing PMNs to then have an

amplified responses to pathogens6,7. There are many types of priming agents, including

fragments secreted by microbes (LPS), chemokines (IL-8), and pro-inflammatory cytokines

(tumor necrosis factor $)7. Paradoxically, primed PMNs can cause more damage to

23

endothelial cells than non-primed PMNs8, which then leads to neutrophil-mediated tissue

injury9. The invasion of primed PMNs, which are hyperactive, is probably one of the leading

causes of acute respiratory distress syndrome (ARDS)9. Furthermore, lung damage has also

been associated with remote organ dysfunction such as epithelial cell apoptosis in

kidney10,11. As the lungs receive the entire cardiac output, it has been hypothesized that they

may play a role in de-priming PMNs, and then release them back to the circulation in a

quiescent state, in order to protect the host tissue from damage caused by primed PMNs9,11.

There has been significant research in the area of neutrophils and lung inflammation

to gain a better understanding of the underlying mechanisms of systemic inflammation. For

pulmonary inflammation, cells can be obtained from bronchoalveolar lavage (BAL), whereby

sterile saline solution (300 mL) is instilled into the lung in aliquots (25 mL) followed

immediately by aspiration3,12 . Sputum from lung tissues13 can also be collected non-

invasively. However, sputum contains mucus, cells and debris, making it harder to be

analyzed by flow cytometry and requiring a precise gating strategy14. Upregulation or

downregulation of specific membrane markers, such as CD11b and CD62L, respectively, has

been used to assess the activation state of PMNs collected from patients with chronic

pulmonary disease3,4,15. An increased number of PMNs extravasated in lung tissue have been

observed in patients with chronic obstructive pulmonary disease (COPD). These PMNs

exhibited high levels of CD11b/18 (Mac-1), suggesting that these PMNs have been primed4.

It is also essential to examine the phenotype of PMNs after extravasation under non-

pathological conditions to determine if the change in expression of membrane receptors is

induced by inflammation in the tissue rather than the extravasation. When characterizing the

phenotype of neutrophils before and after transmigration in healthy individuals and people

24

with disease, Fortunati et al. found that PMNs collected from lung tissues exhibited an

activated phenotype (high CD11b expression and low CD62L expression) under

inflammatory (patients with disease) and non-inflammatory conditions (healthy

participants)3. The difference in neutrophil phenotype could only be identified following

stimulation. Unlike the BAL PMNs of sarcoid patients, the expression of CD32 and CD11b

on BAL neutrophils from healthy participants could not be further upregulated upon fMLP

stimulation3. Altogether, these results suggest that the activated phenotype observed in lung

PMNs is not due to the inflammation but rather a hallmark of extravasation to the lungs, and

that, under healthy conditions, lung neutrophils have an impaired functionality, which may

be due to a protective/suppressive property of the lungs3,16. Upon inflammation in the lungs,

an imbalance may occur in the environment, leading the PMNs to be able to respond to

stimuli, which will in turn lead to lung damage. These results emphasize how the

environment may affect neutrophil phenotype and the importance of assessing neutrophil

response to stimulation.

3.3. Neutrophils in the placenta

Many years ago, it was thought that pregnancy was associated with immune

suppression, which caused both the mother and the fetus to be susceptible to infectious

diseases17. However, the discovery of the presence of several types of immune cells in the

human decidua (part of the endometrium)17 led to a conclusion that the maternal immune

system plays an important role in the fetal-maternal immune adjustment and protection

against pathogens17,18. Neutrophils from term and preterm neonates also behave differently,

whereby the latter showed lower capability of phagocytosis and lower amount of reactive

oxygen species produced19, suggesting that neutrophil phenotype may adapt to various

25

conditions.

The study of maternal, cord blood and placenta has allowed researchers to identify

two neutrophil subsets with different phenotypes - low-density granulocytes (LDGs) and

normal-density granulocytes (NDGs)20. The cells were isolated following various density-

gradient centrifugation procedures. NDGs and LDGs phenotypes were characterized using

flow cytometry with the following markers20:

- CD66b, upregulated on cell membrane upon degranulation,

- CD15, involved in stimulation of degranulation and respiratory burst,

- CD16, involved in degranulation as it is cleaved from the cell membrane when

elastase is released from granules (downregulation upon release),

- CD63, upregulated on cell membrane following release of azurophilic granules.

When compared to NDGs, LDGs expressed higher levels of CD15, CD33, CD63 and CD66b

as well as lower levels of CD1620, suggesting that LDGs had undergone activation and

degranulation (potentially as they were activating to protect the mother and foetus from

pathogens). These results highlight that various phenotypes of neutrophils may co-exist and

the significant potential of flow cytometry to identify if neutrophils have undergone

activation in vivo.

3.4. Neutrophils in the mouth

To maintain an healthy oral environment, the presence of PMNs in the oral cavity is

essential to eliminate ongoing exposure to bacteria21. It is well known that, upon exposure to

bacteria, neutrophils can release reactive oxygen species (ROS) and enzymes and that these

toxic substrates kill not only pathogens but also cause damage to host tissues7,22. The PMNs

in the oral environment are thus exposed to a unique context, which is referred to as para-

26

inflammation, whereby PMNs have the potential to respond to low-grade foreign pathogens

without clinical signs of inflammation and damage to the tissue23. On the other hand, chronic

periodontitis (CP), which is induced by bacteria and biofilm formation, is a destructive

inflammation causing irreversible damage to the periodontium and tooth-supporting

tissue22,23, and oral PMNs are believed to play a significant role in this disease.

PMNs from the oral cavities can be collected using a simple and non-invasive rinsing

method, whereby participants rinse their mouth for 30 seconds with an isotonic sodium

chloride solution (5 ml) and then expectorate into a tube21,22. When compared to blood-

circulating PMNs, oral neutrophils are functionally different in terms of phagocytosis

(evaluated using bright field microscopy to detect the presence of intracellular particles)24,

and also exhibit a lower ability to generate ROS upon stimulation (evaluated by flow

cytometry)22.

Not only has excessive PMNs recruitment been observed in severe or chronic

periodontitis (CP) with a six-fold increase in PMNs counts compared to healthy controls21,25,

but two distinct phenotypes in oral PMNs, para- and pro-inflammatory, have also been

identified in healthy individuals versus individuals with CP, respectively23. The pro-

inflammatory PMNs found in the mouth of CP patients showed elevated degranulation,

phagocytosis, ROS production, and neutrophil extracellular traps (NETs) release23.

Furthermore, studies of ROS generation in oral neutrophils from individuals with refractory

periodontitis also identified the existence of two groups: low (LR) and high responders

(HR)22. HR showed a more substantial difference between resting and stimulated ROS

levels compared to the difference in levels observed in LR, suggesting that HR hold a more

significant activation potential than LR and such an enhanced response to stimuli may result

27

in more destructive tissue damage with periodontitis in HR versus LR22.

Interestingly, para-inflammatory PMNs in the healthy oral cavity can be further

divided into two subpopulations, para1 and para2, as defined by Fine et al23. Compared to

para1, the para2 population is more activated, showing increased expression of activation

markers (CD55 and CD63), and decreased expression of a phagocytosis marker (CD16)23.

The two para-inflammatory populations appear to co-exist in the healthy oral cavity

environment, with no tissue damage induced by the para2 (intermediate activated)

population. In chronic periodontal diseases, only the PMNs present exhibit the pro-

inflammatory phenotype, which result in the continuous damage to the periodontium. It is

yet unclear what causes the shift in phenotype, but the presence of biofilm and the locally

inflamed oral environment may provide molecular cues to oral PMNs in the mouth23.

Studies of PMNs that have extravasated to the oral cavity in healthy or inflamed/infected

environment not only highlight the complex nature of neutrophil phenotypes but also how the

state of the local environment may induce one phenotype over another.

3.5. Neutrophils in the eye

3.5.1. Ocular Immune Privilege

The brain, the eye, the uterus, and the testis are all immune privileged sites in our

body26. Immune privilege was thought to be “immune ignorance”, which means completely

blocking the entry of immune cells to the tissue26. However, this simplistic view of immune

privilege poorly represents the dynamic mechanisms involved27. Shechter et al proposed

that “the privilege of these organs resides not in their ability to block passive immune

infiltration or to facilitate active immune tolerance or ignorance, but rather in the ability of

the epithelium gate system to orchestrate active communication with the circulating immune

28

system”26.

The eyes continuously encounter external threats, such as microbes, pollen and

allergens, but the incidence of immune-mediated blindness is relatively rare due to immune

privilege27. However, the presence of biomaterials, for example contact lenses, may

challenge the immune privileged ocular environment and contribute to the development of

adverse events, such as microbial keratitis and contact lens-associated corneal infiltrates28.

Investigating neutrophils, the first responder innate immune cells, is therefore important to

gain a better understanding on their contribution to ocular inflammation and interactions with

ocular biomaterials.

3.5.2. Overview of the ocular environment

The eye is a sensory organ that is responsible for vision. It is enclosed by three layers

of tissue, the innermost retina, the middle uveal tract, and the outermost sclera and cornea29,

as shown in Figure 5. Light rays enter the eye through a transparent tissue, the cornea, and

the intensity of the light is adjusted by the iris muscles through contraction. Then, through a

process called accommodation, the lens focuses the light rays on the retina which is

embedded with photoreceptor cells, rods and cones29. The optic information is then sent to

the brain via the optic nerves.

29

Figure 5 - Anatomy of the human eye30. Reprinted with permission from: Kels, B. D., Grzybowski, A. & Grant-Kels, J. M. Human ocular anatomy. Clin. Dermatol. 33, 140-146 (2015). © Elsevier 2015.

3.5.3. Tear film and its components

The human eye is directly exposed to the external environment (which contains

various pathogens31), which may lead to eye diseases, such as microbial keratitis (MK). MK

is one of the most common corneal infections caused by microorganism that could result in

vision loss32. It is thus essential to have a structure, the tear film, acting as a barrier between

the eyes and the outer environment31 to reduce this occurrence. Components of the tear film

are secreted by different epithelial and glandular tissues, such as the meibomian glands (the

lipid layer), lacrimal glands (the aqueous layer), and the goblet cells within the conjunctiva

(the mucin layer)33. The tear film was initially thought to have three layers, the innermost

mucin layer, the middle aqueous layer and the outermost lipid layer34, as shown in Figure 6a.

This was further modified, with the lipid layer divided in the outermost non-polar phase

exposed to the air, and the polar phase adjacent to the aqueous layer35, as illustrated in Figure

30

6b. The mucin layer is in direct contact with the cornea, and forms a gel-like structure which

helps to hold tears and provides a wet surface for blinking31. The aqueous layer lubricates

the surface of the eye when blinking31. This layer contains aqueous contents secreted by the

lacrimal gland and also numerous proteins, such as lysozyme and lactoferrin31, which

provide nutrition to the cornea and protect the eye against pathogens31. The polar phase

keeps the ocular surface moist, while the non-polar phase acts as a barrier and also helps to

relieve the surface tension31,33,36.

Figure 6 – a) An old view of three layered tear-film and b) the polar and non-polar phases within the lipid layer 31. Reprinted with permission from: Cwiklik, L. Tear film lipid layer: A molecular level view. Biochim. Biophys. Acta - Biomembr. 1858, 2421–2430 (2016). ©Elsevier2016.

However, the layers of tear film have been revised. Instead of having the three border

layers, it has been proposed that the tear film consists of the outermost non-polar lipid layer,

the polar lipid layer, the aqueous-mucin layer, and a glycocalyx layer covering the cornea35,

as shown in Figure 7.

31

Figure 7 - The modern view of the tear film37. Reprinted with permission from: Green-Church, K. B., Butovich, I., Willcox, M., et al. The International Workshop on Meibomian Gland Dysfunction: Report of the Subcommittee on Tear Film Lipids and Lipid–Protein Interactions in Health and Disease. Invest Ophthalmology Vis Sci. 52, 1979 (2011). © Association of Research in Vision and Ophthalmology 2011.

To function optimally, tear film stability is important, or its disruption may result in

dry eye. Evaporation rate has been shown to be one of the major factors affecting the

stability of the tear film38. There is a strong correlation between the thickness of the lipid

layer and the evaporation rate38,39, which means “the thicker the lipid layer, the less

evaporation and more stable the tear film”33. Therefore, the lipid layer plays a crucial role to

maintain the normal function of the tear film36.

Zhou et al. identified 1543 tear proteins in tears from healthy subjects40, with

lysozyme, lactoferrin, tear lipocalin and secretory immunoglobulin A (sIgA) being the main

proteins found in the tear fluid41,42. Aside from the above listed proteins, tears contain

32

cytokines, which are small proteins that are important in the recruitment and activation of

immune cells, as well as growth factors. Cytokines have been suggested to play an essential

role in corneal homeostasis43. Research also showed that the components of the open-eye

and closed-eye tears are significantly different from each other44,45. Closed-eye tears contain

higher levels of albumin, fibronectin, complement proteins, sIgA and plasmin compared to

open-eye tears44–46, with sIgA representing up to 80% of the total protein content45.

Furthermore, a large number of neutrophils have been observed in closed-eye tears45.

3.5.4. Tear-film neutrophils

Neutrophils have been observed on the ocular surface in the absence of a wound or

infection and have recently been termed “tear-film neutrophils” or “tear-film PMNs” to

reflect their location45,47,48. Recent studies have collected tear-film neutrophils during the

day time as well as following closed-eye conditions. While up to a few thousands PMNs can

be collected from open-eye tears49, hundreds of thousands to millions of PMNs have been

collected after sleep (closed-eye conditions)48.

The origin of tear-film PMNs has yet to be determined. Two hypotheses have been

presented40,48,50, one is extravasation, whereby chemotaxis leads to their migration to the

ocular surface from conjunctival blood vessels; the other is that neutrophils are present in

lacrimal glands, and are brought to the ocular surface with the tear fluid from the lacrimal

glands. In the latter, the hypothesis is that in the closed environment, there is limited

replenishment and blinking and thus tear-film PMNs accumulate. Chemokines play an

essential role in the recruitment of PMNs to local tissues. Hence, the recruitment of tear-film

PMNs to the ocular surface may be induced by the presence of specific cytokines (e.g., IL-8)

and complement proteins (e.g., C5a), which have been shown to be present in the closed-eye

33

environment in large amounts51. The presence of IL-8 and C5a, chemoattractant factors for

PMNs, was recently characterized in tear fluid (TF) samples collected at three time points,

before (evening TF) and after sleep (morning TF), and around noon (noon TF)52.

Concentrations of IL-8 and C5a in the morning TF (after sleep) were the highest compared to

the noon TF52. Interestingly, tear fluid collected in the evening contained a high level of

C5a, suggesting that the complement system is probably activated, which may due to the

accumulation of microorganisms and dust particles, prior to eye closure52. Both human

corneal epithelial cells (hCECs) and PMNs can secrete IL-8 in response to the presence of

microorganisms and dust particles, and this may also contribute to the high levels of IL-8

observed in the morning TF53,54. Hence, the activation of complement pathway before eye

closure may lead to the recruitment of PMNs, which could be further promoted by the

increased level of IgA. IL-8 secreted by hCECs mediates PMNs migrating to the eye, and

this recruitment may be further enhanced with the co-secretion of IL-8 by both hCECs and

PMNs. Low levels of L-selectin were also observed on tear-film neutrophils collected after

sleep, which may have been lost during extravasation48. As mentioned earlier, low

expression of L-selectin has been observed in lung PMNs 3 and oral PMNs23, which have

already been proven to come from the blood vessels. Collectively, results from high levels of

chemoattractant in evening TF and shedding of L-selection on collected tear-film PMNs

strongly suggest that tear-film neutrophils present in the closed-eye environment have

extravasated and come from the conjunctival blood vessels.

3.5.5. The non-inflammatory phenotype of tear-film PMNs

The phenotype of tear-film neutrophils collected from the closed-eye environment has

just started to be characterized. Compared to blood circulating PMNs, tear-film PMNs

34

collected from the eyes after sleep have been shown to have a significantly higher expression

of CD66b and lower expression of CD62L48,52,55. One research group reported that there is

no difference in CD11b expression between tear-film and blood-isolated PMNs52, whereas

two other groups demonstrated that the level of CD11b is higher in tear-film PMNs48,55. In

addition, tear-film PMNs are unable to upregulate key markers of activation upon

stimulation, such as with phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS)

and N-Formylmethionyl-leucyl-phenylalanine (fMLP)48,55, suggesting that they may have a

non-inflammatory phenotype or may have been activated in vivo and are unable to respond to

further stimulation. To better understand the phenotype observed with tear-film PMNs,

experiments were performed by Postnikoff and Gorbet to expose blood-isolated PMNs to

conditions similar to the closed-eye environment56. Results showed that in vitro exposure to

hypoxic conditions, hCECs and artificial tear solution cannot induce the non-inflammatory

phenotype in blood-isolated PMNs56.

Postnikoff and Nichols hypothesized that tear-film PMNs have already been activated

as they arrived at the ocular surface due to the reduced expression of CD14 and CD1655.

CD14 and CD16 are markers for phagocytosis57,58, so the decrease in expression is believed

to be associated with the reduced phagocytic capability of PMNs. This observation appears

to correlate with results indicating that PMNs recovered from the closed-eye tears may have

an impaired phagocytic ability59. Although reduced levels in CD16 is also related to

apoptosis60, tear-film PMNs were not reported to be apoptotic48. CD43, which acts similarly

to CD16, was used by Mahajan et al. to characterize the tear-film PMNs, and results showed

that tear-film PMNs expressed low level of CD4352. This decrease in the expression of

CD43 is associated with the cleavage by the neutrophil elastase after phagocytosis or

35

stimulated NADPH oxidase61–63, indicating tear-film PMNs have already degranulated. In

addition, eye discharge collected after sleep contains PMNs, NETs, dead epithelial cells and

entrapped bacteria52, suggesting that tear-film PMNs have the capability to eliminate

pathogens when they are extravasated to the ocular surface by releasing NETs, phagocytosis

and degranulation. Taken all together, those findings suggest that tear-film PMNs might

have undergone activation during the sleep.

Interestingly, tear-film PMNs at both 1-hour and 7-hour time point showed no

difference in their lack of ability to respond to inflammatory stimuli (LPS and CaI)55, which

would tend to suggest that their non-inflammatory phenotype is independent of their

residence time in the ocular environment.

3.5.6. Tear-film PMNs and ocular surface diseases

As neutrophils are innate immune response cells, their role in other systematic

inflammation, such as sepsis and acute respiratory distress syndrome (ARDS), has been

largely investigated, but remains largely unknown in the ocular inflammation. Due to the

improved collection protocols that have been established, enough tear-film PMNs can be

collected from the ocular surface, allowing for the subsequent phenotype-characterizing

experiments. This has led tear-film neutrophils to be a new area of research in ocular surface

inflammatory diseases.

Dry eye disease is a common ocular complication which is caused by insufficient

generation of tears or the quick evaporation rate of tears39. According to several findings, T

cell-mediated inflammation is considered to be associated with dry eye disease (DED)64,65.

However, the role of PMNs in DED remains undefined and several recent studies point

towards a potential role of these innate immune response cells. Significantly more PMNs are

36

found in the tear samples of meibomian gland dysfunction (MGD) patients compared to

healthy people, and a positive correlation exists between the number of PMNs and the

severity of MGD66. Furthermore, an excessive amount of molecular components of NETs

have been observed on the ocular surface of severe DED patients, and similarly to MGD, the

amount of NETs has been positively correlated with the severity of DED67. The continuous

formation of NETs by PMNs is believed to be induced by hyperosmolar stress, one of the

core mechanisms of DED67. Due to the absence of nuclease, which is involved in the

clearance of extracellular DNA, NETs accumulate on the ocular surface and may lead to the

ocular inflammation observed in DED67,68. Postnikoff et al. also showed that twice as many

PMNs are collected from the DED patients upon awakening (after sleep) compared to

healthy individuals, but there is no difference in the numbers of PMNs in the open-eye

tears49. People with DED also have a higher neutrophil-lymphocyte ratio, approximately

64:1 upon awakening compared to 21:1 in healthy eyes, suggesting that tear-film PMNs may

affect T cells recruitment49.

Ocular allergy (OA) is a general term that includes the two common diagnoses,

seasonal and perennial allergic conjunctivitis (SAC and PAC, respectively)69. The

underlying mechanism of SAC and PAC is mediated through IgE causing hypersensitivity

reactions to allergens69. SAC occurs mainly in the spring and summer due to the presence of

airborne pollens, whereas PAC can occur throughout the year when exposed to perennial

allergens69. Itchy eyes, tearing, redness and sensitivity to light are common symptoms that

affect over 30% of the North American population suffering from seasonal allergies70.

Eosinophils, which release cytokines upon activation, are a hallmark of an allergic response71

and have been observed on the ocular surface72. Several studies on seasonal ocular allergies

37

have shown an increased level of cytokines and also indicated the presence of leukocytes in

tears71,73–75. However, no study has yet characterized the leukocyte population and its

phenotype in the tear-film of individuals suffering from ocular allergies.

These recent findings and observations suggest that tear-film PMNs may have a role

in ocular surface diseases. It is thus important to characterize their phenotype to enable

future development of diagnostic and therapeutic strategies.

3.6. Conclusion

From the blood to the mouth and other tissues where neutrophils are found, it is

evident that the neutrophil population is heterogeneous, and different phenotypes exist in

both physiological and pathological conditions. Tear-film neutrophils represent a novel

research avenue and there is currently limited knowledge as to their phenotype and origin.

Tear-film neutrophils collected from the closed-eye environment appear to be non-

inflammatory and the reduced expression in L-selectin suggest that they have extravasated

from the conjunctival blood vessels. Similarly, to PMNs found in the lung, placenta and

mouth, tear-film PMNs have a high expression of CD11b and CD66b but low expression of

CD16 compared to blood-isolated PMNs, suggesting an already activated phenotype.

However, unlike PMNs collected from the lung, placenta, and mouth, the involvement of

tear-film PMNs in ocular diseases remain largely unknown as does ROS generation. There is

a need for a better understanding of the mechanisms involved in impaired functionality and

interaction with other immune cells. To support this need, it is also important to develop

reliable protocols for collection and immunophenotyping and further characterize the

phenotype of tear-film neutrophils in healthy individuals.

38

Chapter 4

Effects of Experimental Procedures on The Expression of Membrane

Receptors on Tear-film Neutrophils

4.1. Introduction

Neutrophils (PMNs) are essential inflammatory cells of the innate immune system1,

and are the first cells to arrive at infected sites2. It is thought that, under normal

physiological conditions, recruitment of immune cells to the ocular surface is restricted due

to immune privilege3,4. However, despite the immune privilege conferred to the eye, a

significant infiltration of neutrophils occurs on the ocular surface when eyes are closed for a

prolonged time (such as during sleep)4–6; these cells are often referred to as tear-film

neutrophils from the closed-eye environment4. As cells of the innate immune system with a

significant role in the inflammatory response, their presence on the ocular surface has

generated significant interest, since many of their roles and functions in the closed eye

environment have yet to be discovered.

Neutrophils, which circulate in blood, have been well characterized, and recent

research indicates that the neutrophil population is heterogeneous, as PMNs found in

different types of tissues, such as the lungs7, the mouth8, the placenta9 and the eye4, have

been shown to exhibit distinct phenotypes. To develop a better understanding of these

neutrophil populations, functionality studies have been carried out to assess cell

activation4,10,11, which can effectively be characterized by flow cytometry via measuring the

expression of cell membrane molecules12,13. Among others, CD11b, CD16, CD55 and

CD66b are common membrane receptors used to characterize leukocyte activation and

39

phenotype. CD11b, also known as the macrophage-1 antigen (Mac-1) or complement

receptor (CR3), plays a crucial role in PMNs transmigration and mediating

immune/inflammatory response and adhesion14. The upregulation of CD11b occurs early in

the cell activation process2 and is often used as an indicator for cell activation and adhesion15.

CD16, a member of the Fc gamma receptors for immunoglobulin, is shed from the cell upon

neutrophil elastase release, and has been used as a phagocytosis and degranulation

marker16,17. CD55 (decay-accelerating factor), a glycosylphosphatidylinositol (GPI)-

anchored receptor and regulator of complement activation, is mainly stored in secretory

vesicles and is upregulated on the cell membrane after activation18. CD66b is recognized to

be a marker for degranulation, and its expression is known to increase upon activation15.

In immunophenotyping experiments, fixation is often involved to preserve cell

structure and avoid undesired cell activation and any artefactual changes. Fixation also

contributes to preserving proteins, carbohydrate and other bio-active substances and prevents

cell decomposition, putrefaction and autolysis19,20. Aldehydes, including paraformaldehyde

(PFA) and formaldehyde21, are the fixative agents most commonly used and preserve cell

structures by combining the nitrogen base in one protein with an already formed

hydroxymethyl group in another protein, and forms a crosslink methylene bridge (CH2)19.

PFA is most commonly used in flow cytometry and the fixation process of blood cells,

especially lymphocytes, has been thoroughly investigated. There are two ways to fix cells,

(1) adding PFA before the staining with antibodies (pre-fixed staining) and (2) adding PFA

after the staining with antibodies (post-fixed staining). Fixation prior to staining has been

shown to significantly decrease the expression of CD11b, CD18 and CD62L on blood

leukocytes22. It has also been observed that expression of CD16 on blood-isolated PMNs

40

gradually decreases with increased time of post-fixation23, suggesting that a fixation step can

significantly affect the expression of the marker of activation being investigated. As

heterogeneity exists in neutrophil population, it is not known if similar effects of PFA would

be observed on the expression of membrane receptors on tear-film PMNs.

Neutrophils are also delicate and fragile cells that can be easily damaged by

inappropriate experimental procedures24. For example, counterflow centrifugal elutriation, a

protocol used to isolate PMNs, has been shown to cause an increased release of superoxide

and granule contents when PMNs are activated25. Mechanical forces during centrifugation

and resuspension as well as exposure to warm or cold temperature (37oC versus 4 oC) result

in changes in the expression of C3b receptors (CD35) and CR3 (CD11b) on PMNs26,27.

Since tear-film PMNs have a distinct phenotype from blood-isolated PMNs, it is likely

unreasonable to assume that tear-film PMNs will be impacted in the same manner as blood

leukocytes. Experimental procedures have the potential to significantly affect results and

have been largely unexplored with tear film PMNs. It is however a key component to ensure

reproducibility and reliability of results. The objective of this study was thus to determine if

experimental procedures, such as centrifugation and fixation, could alter the expression of

cell activation membrane receptors. Using flow cytometry, the expression of CD11b, CD16,

CD55, and CD66b on tear-film PMNs were characterized in unfixed samples and compared

to pre-fixed stained (adding PFA before staining with antibodies) and post-fixed stained

(adding PFA after staining with antibodies) and the centrifuged samples. Changes in the

expression of CD45, the common leukocyte marker, were also assessed.

41

4.2. Materials and Methods

4.2.1. Reagents and Monoclonal Antibodies

Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against human

CD11b and CD66b, R- phycoerythrin (PE)-conjugated monoclonal antibodies against human

CD55 and CD16 and R-phycoerythrin-cytochrome 5 (PE-Cy5)-conjugated monoclonal

antibody against CD45 were purchased from Becton Dickinson Pharmingen (San Diego, CA,

USA). Paraformaldehyde (PFA) was from Sigma-Aldrich Co. (Oakville, Ontario, Canada).

Phosphate-buffered saline (PBS) was purchased from Lonza (Allendale, New Jersey, USA).

Dulbecco’s modified eagle media (DMEM) was purchased from Life Technologies

(Burlington, Ontario, Canada). Keratinocyte serum-free medium (KM) was purchased from

ScienCell (Carlsbad, California, USA).

4.2.2. Cell Collection

This study was conducted in accordance with the tenets of the Declaration of Helsinki

and received ethics clearance from the University of Waterloo Human Research Ethics

Committee (#30164; Waterloo, ON, Canada). A total of five healthy participants, with an

age ranging from 23 to 30, were involved. Each participant provided one or more samples

for each experiment.

Participants were provided with instructions and were trained to self-collect tear-film

neutrophils after sleep using a polypropylene pipette containing sterile PBS. They slept at

home, as usual. After 8 hours of sleep, upon awakening, participants gently washed their

eyes with approximately 5 mL of PBS for each eye; the eye wash was collected in one sterile

polypropylene tube (pooled sample) (Figure 8)4. After collection from both eyes, the tube

42

was placed in the provided storage container and delivered to the lab within two hours.

Figure 8 - Illustration of the at-home tear-film PMNs collection process. The required materials, 1) empty polypropylene tube, 2) disposable pipette sterile, and 3) phosphate buffered saline (PBS), are shown in (a). The overall demonstration of the collection procedure is shown in (b). It is recommended that the procedure be performed in front of the mirror. Participants used the polypropylene disposable pipette 2) to draw approximately 1 mL of PBS 3) and the pipette containing PBS was put at the inner angle of the eye with caution and as the pipette bulb is squeezed, PBS is slowly released and flows onto the ocular surface. The runoff was collected at the outer angle of the eye by using an empty polypropylene tube 1) Participants used approximately 5 mL of PBS per eye.

4.2.3. Experimental procedure

Once cell samples were delivered to the lab, they were centrifuged at 290xg for 10

min and resuspended in approximately100 µL of PBS. Cell count and viability were

determined using trypan blue. Samples were diluted to a final cell concentration of 100,000

cells/mL in an artificial tear solution (keratocyte medium containing various tear film

proteins), as detailed in Table 2.

43

Table 2 – Components of the artificial tear solution

Components Mass (mg)

Albumin 2

IgG 0.2

Lactoferrin 18

Lysozyme 19

Mucin 1.5

KM 10mL

KM: keratocyte medium. KM is endotoxin-free, sterile and contains various salts and amino acids and has previously been used as a tear substitute for in vitro studies.

Our control samples were the cell suspension that was processed immediately for

flow cytometry without any incubation and is referred to as the “time zero” samples (T0

samples). To determine the impact of re-exposure to physiological temperature, cell samples

in polypropylene tubes were incubated for four hours in a cell incubator (37oC, 5% CO2).

These samples are referred to as “4-hour incubation” samples (4h samples). Each T0 and 4h

sample was also divided into centrifuged and non-centrifuged groups. Centrifuged groups

were spun down at 280xg for 5 min and then re-suspended in PBS. The non-centrifuged

group stayed on the bench without any involvement of centrifugation. Immediately following

centrifugation, all samples were processed for flow cytometry.

4.2.4. Fixation

To avoid confounding factors of experimental procedures (centrifugation, exposure to

change in temperature, etc), the impact of fixation on the expression of membrane receptors

was assessed on T0 samples only. The effect of fixation was examined as follows: pre-fixed

staining, post-fixed staining and no fixation.

For pre-fixed staining samples, a small aliquot of cell suspension was transferred into

44

different labelled tubes containing 1:1 DMEM/10% FBS and 2% PFA (1% final

concentration) followed by incubation at 4oC for 15 min, the common protocol for pre-

fixation of samples22,28. After fixing, fluorescently-labelled antibodies against CD11b, CD16

CD45, CD55, and CD66b were added to corresponding tubes and samples were incubated for

20 min in the dark. Following incubation with antibodies, samples were diluted by adding

DMEM/10% FBS and stored in the fridge until flow cytometry analysis the next day.

For post-fixation staining, the usual procedure for immunostaining was followed,

whereby a small aliquot of cell suspension was incubated in tubes containing DMEM/10%

FBS and fluorescently-labelled antibodies against CD11b, CD45, CD55, CD66b, or CD16.

Samples were then incubated for 20 min at room temperature in the dark. At the end of

incubation, samples were diluted with DMEM/10% FBS and fixed with PFA (1% final

concentration). Samples were then stored in the fridge until flow cytometry analysis the next

day.

For the unfixed samples, the procedure remained the same as the post-fixation

samples except in the last step, PFA was replaced by DMEM/10% FBS. The unfixed

samples were analysed by flow cytometry within 15 min.

The experimental procedure for the samples is illustrated in Figure 9. Note that due

to the effect of pre-fixation (see results), all 4h samples were processed using the post-fixed

staining method.

45

Figure 9 - Experimental flow chart. After cell collection, cells were divided into two groups, time zero samples were processed right after cell count (T0), and 4h samples were incubated for 4 hours at 37 oC (4h). Cells from T0 and 4h groups were either centrifuged or not, and T0 non-centrifuged group was used to examine the impact of PFA. The rest of the groups, T0 centrifuged, 4h centrifuged, and 4h non-centrifuged were fixed after staining with antibodies.

4.2.5. Flow cytometry

All samples were acquired on a Becton Dickinson FACSCalibur flow cytometer

(Mountain View, CA, USA) using CELLQuest Software (Becton Dickinson, Mountain

View, CA, USA). At least 2000 PMNs events were acquired, and fluorescent intensities were

recorded as arbitrary fluorescent units (AFU). The PMNs population was identified by

doubled-gating on size and CD45+ cells 4. In order to compare expression of membrane

receptors between different conditions, the ratio of fluorescent intensities of non-centrifuged

and non-fixed samples (baseline) versus other conditions are presented.

4.2.6. Statistics

All results are reported as mean ± standard deviations. To assess the significant

differences in the ratio of fluorescent intensities, paired sample t-test and Wilcoxon signed-

46

rank test were performed using IBM SPSS Software, and a p-value less than 0.05 was

required for statistical difference.

4.3. Results

On average, the number of leukocytes collected by using the gentle eye wash method

was 2.2 x106 (ranging from 1.1x106 to 9.4 x106 leukocytes) with less than 2% dead cells. The

effect of fixation, centrifugation and incubation were assessed on the expression of CD11b,

an activation and adhesion marker, CD16, a degranulation and phagocytosis marker, CD45, a

common leukocyte marker, CD55, a complement activator marker, and CD66b, a

degranulation marker, on tear-film PMNs.

4.3.1. The impact of fixation

As shown in Figure 10, pre-fixed and post-fixed staining affected the expression of

activation markers on tear-film PMNs in different ways when compared to the non-fixed

staining samples. When PFA was added prior to staining with antibodies, a significant

decrease (78 to 98 % reduction) was observed in the expression of CD11b (p <0.001), CD16

(p = 0.008), and CD45 (p <0.001). Conversely, the expression of CD55 and CD66b

remained relatively unchanged (p ≥0.84). On the other hand, in the post-fixed staining

group, a reduction of more than 50% was observed in the expression of CD16 (p = 0.008)

and a small (14%) but statistically significant reduction in expression of CD11b (p = 0.01).

The other markers, CD45, CD55 and CD66b, on post-fixed staining samples displayed some

variations in expression but remained relatively unchanged (p ≥ 0.36). The results indicated

that exposing collected cells to PFA prior to staining with antibodies resulted in drastic

reductions in CD11b, CD16 and CD45 expression and thus for the remainder of the study,

47

cells were fixed post staining.

Figure 10 - Effect of PFA on the expression of cell activation surface markers on tear-film PMNs. Cells were either fixed with 2% PFA at 4oC for 15 min prior to staining with antibodies (pre-fixed staining) or stained with antibodies and then fixed with 2% PFA (post-fixed staining). Control samples were stained with antibodies and immediately analyzed by flow cytometry (non-fixed staining). All incubation with antibodies were performed at RT, in the dark, for 20 min. Fluorescence intensities for CD11b, CD16, CD45, CD66b, and CD55 were recorded as the arbitrary fluorescent units by flow cytometry and are expressed as a percentage of expression level compared to the non-fixed staining samples (resting samples). The dotted line represents the resting value. Mean ± SD, *significantly different from non-fixed samples, p < 0.01.

48

4.3.2. The impact of incubation temperature

To assess how exposure to physiological temperature following collection may

activate tear-film PMNs, cells were exposed to a 4-hr incubation at 37oC, 5% CO2 and

expression of cell activation membrane receptors was evaluated by flow cytometry and is

shown in Figure 11. When compared to T0 samples, exposing collected tear film PMNs to

37oC for 4 hours appeared to induce a mild state of cell activation, with a significant (p =

0.04) upregulation in CD11b (~ 30% increase) and CD45 (~ 50% increase) while expression

of CD16 and CD66b decreased markedly; however, due large variations in response, the

more than 30% reduction in CD16 and CD66b expressions were not statically significant (p

= 0.09 and p = 0.06, respectively). The reduction in CD66b was also inconsistent with the

lower expression of CD16, an indicator of degranulation that is cleaved upon degranulation;

CD66b expression is expected to increase with release of granules. CD55 expression

remained mostly unchanged (p = 0.78).

49

Figure 11 – Effect of exposure to 37oC for 4h on the expression of cell membrane activation markers on tear-film PMNs. Following collection, tear film PMNs were either processed for flow cytometry (antibody staining followed by PFA fixing) or incubated for 4hrs at 37oC, 5% CO2. Following 4hr-incubation, cells were stained with antibodies and fixed with PFA. Fluorescence intensities for CD11b, CD16, CD45, CD66b, and CD55 were recorded as arbitrary fluorescent units by flow cytometry and are expressed as a percentage of expression level compared to the time zero samples (resting samples) (T0). The dotted line represents the resting value (T0). Mean ± SD, *significantly different from non-incubated samples, p < 0.05.

50

4.3.3. The impact of centrifugation

To determine the effect of centrifugation on the expression of cell membrane

activation markers, tear-film PMNs from T0 and 4h-incubated groups were either centrifuged

at 280xg (5 min) or not. Changes in the expression of cell activation membrane receptors are

reported in Figure 12. For T0 samples, results indicated that an extra centrifugation step,

which exposed cells to mechanical forces, caused some degranulation, as a significant

decrease in the expression of CD16 (p <0.001) and an increase in CD66b (p = 0.16) were

observed. Reduction in the expression of CD45 (p <0.001) and CD55 (p = 0.07) also

occurred. The expression of CD11b was not affected by centrifugation (p = 0.22).

For the 4h samples, all changes observed with the time zero samples were further

amplified after the 4hr incubation at 37oC, confirming our above results that cells were

affected by the 37oC incubation (in section 4.3.2, we had identified signs that tear film PMNs

may have undergone some degranulation) and that exposing cells to centrifugal mechanical

forces induces further cell damage and degranulation. The expressions of CD11b (p = 0.03),

CD16 (p = 0.003), CD45 (p <0.001) and CD55 (p = 0.004) were significantly reduced

while, as expected from a degranulation process, the levels of CD66b (p <0.001)

significantly increased.

51

Figure 12 – Effect of centrifugation on the expression of cell membrane activation markers on tear-film PMNs. Following collection and concentration, tear film PMNs were either subjected to another centrifugation step or incubated for 4 hrs at 37oC – 5%CO2 and then centrifuged. Samples were processed for flow cytometry (antibody staining followed by PFA fixing). Fluorescence intensities for CD11b, CD16, CD45, CD66b, and CD55 were recorded as arbitrary fluorescent units by flow cytometry and are expressed as percentage of expression level compared to the non-centrifuged time zero samples (resting samples). The dotted line represents the resting value. Mean ± SD, n = 8 – 10, *significantly different from resting, p < 0.05.

52

4.4. Discussion

As summarized in Table 3, our results identified how cell membrane activation

receptors exhibit different sensitivity to experimental conditions (centrifugation, exposure to

37oC, fixation). For example, minimal changes occur in CD55 expression with fixation and

37oC incubation, while CD16 expression, a marker for degranulation, was the most sensitive

to all experimental procedures tested leading to a significant decrease in expression. Post

fixation was the most reliable fixation step, but an additional centrifugation step appeared to

significantly affect membrane receptor expression (CD11b, CD66b). The level of CD45

remained unchanged only in post-fixed condition, however its changes in relation to

experimental procedures are not as important as it is used mainly to identify leukocytes and

not to assess leukocyte activation. The impact of experimental procedures on tear-film

PMNs antigen expression is further discussed in details below.

Table 3 – Summary of changes in the expression of surface receptors on tear-film PMNs (processed versus unprocessed sample).

CD

markers

Pre-fixed

staining (%)

Post-fixed

staining (%)

Centrifugation

T0 ~ 4 hr sample (%)

Incubation

4hr-37oC (%)

CD11b -85 ± 4 * -14 ± 8 * -(2 ~ 26) ± 14 +28 ± 25 *

CD16 -98 ± 1 * -49 ± 22 * -(58 ~ 70) ±19 -41 ± 31

CD45 -78 ± 5 * +2 ± 23 -(36 ~ 60) ± 10 +52 ±44 *

CD55 0 ± 33 -5 ± 26 -(13 ~ 46) ± 15 0 ± 40

CD66b -4 ± 14 -1 ± 15 +(16 ~ 46) ± 26 -33 ± 24

Changes in expression of surface receptors on tear-film PMNs when exposed to experimental conditions: results obtained from different experiments are summarized. “+” represents an increase while “-” represents a reduction in expression. *Significantly different from rest values, p < 0.05.

53

To further demonstrate the effect of experimental procedures on expression, the

fluorescent values of one participant are presented in Table 4. The low CD16 and high

CD11b and CD66b suggest that tear-film PMNs may have already been degranulated and

extravasated, indicating an activated phenotype of tear-film PMNs, corroborating previous

studies4.

Table 4 - Change in level of expression of receptors on tear film neutrophils following fixation.

CD markers Non-fixed staining Pre-fixed staining Post-fixed staining

CD11b 527 72 422

CD16 67 2 23

CD45 171 33 163

CD55 273 305 226

CD66b 555 529 533

Values are from one participant and are representative from all experiments. Values are reported as arbitrary fluorescent units (AFU).

In flow cytometry, it is crucial to ensure that the preparation procedures before

labelling with fluorochrome-conjugated antibodies exert a minimal effect on antigen

expression. This study aimed to investigate the effect of pre-fixation and post-fixation

staining and centrifugation on expression of membrane receptors on tear-film PMNs in order

to optimize experimental procedures to preserve the expression of markers of activation as

close as possible to the in vivo state by limiting in vitro changes. Any significant change

induced by procedures would significant impact our interpretation of the results on the state

of activation of tear-film PMNs.

Fixation, through the crosslinking of antigens and their antibodies, aims to preserve

the binding structure for a prolonged time. However, research has shown that exposure of

cells to fixative chemicals might cause damage to the expressions of function-associated

54

antigens29, and an increase in autofluorescence12. According to our results, pre-fixed staining

significantly decreased the expressions of CD11b, CD16 and CD45 on tear-film PMNs.

Blood-isolated PMNs and hematopoietic cells have also shown a decreased the expression of

CD11b with fixation prior to staining12,28,29. Fixation prior to staining may lead to a loss of

antigenicity of the epitopes30. Aldehyde fixative reacts with amine groups and forms a

methylene bridge to crosslink between proteins. During this process, the fixative may distort

the tertiary structure of proteins28, and consequently, cause the decrease in recognition of

antibodies to their corresponding antigens. Depending on their structure, antigens may be

affected differently which would explain why a pre-fixation step induced significant damage

on CD11b, CD16 and CD45 but not on CD55 and CD66b. The changes induced by exposure

to PFA prior to staining would have significant impact on our interpretation of results, ie low

CD16 expression would indicate that tear film neutrophils had undergone extensive

degranulation in vivo, although in this case, this is an artefact of how the cells were processed

following collection.

A fixation step following staining significantly reduced the expression of CD16 and

CD11b. Although the 14% downregulation of CD11b compared to the non-fixed sample was

statistically significant, it may not carry biological significance and in this case would not

significantly affect our interpretation of results in the context of cell activation. On the other

hand, as with pre-fixed staining, CD16 warrants caution. A dramatic decrease in the

expression of CD16 was previously reported by Stewart et al. after 24 h fixation on blood-

isolated PMNs, and expression was also observed to further decrease between 24 and 96 h

after fixation23. They also observed a small but significant reduction (12%) in CD11b after

24 h fixation23, which is closed to the 14% reduction in CD11b we observed in our post-fixed

55

samples analysed around 24hrs. Interestingly, we also had observed a change in levels of

expression with some of our activation markers depending on the time of storage after

fixation post staining (results not shown), which is in agreement with Stewart et al.’s

research on blood-isolated PMNs. Thus, to increase result reproducibility, all our samples

were always analyzed around 24 h after fixation. The decrease in level of expression during

4oC storage after fixation may be due to internalization of surface receptors. Fixation tends

to permeabilize cell membranes, resulting in a decrease in cell membrane integrity, which

could eventually cause membrane receptors internalized23,28. The fact that not all receptors

are affected the same way is not surprising, as differences exist in the stability of the antigen

and antibody bond and the linkage between the antibody and its fluorochrome31.

Tear-film PMNs incubated at 37oC for 4 hours showed a significant upregulation in

CD11b and CD45, suggesting activation of tear film PMNs, which would be similar to

previous observation in blood leukocytes32. This upregulation is noteworthy, as CD11b

upregulation on tear-film PMNs has been shown to be minimal upon stimulation with fMLP,

PMA, and LPS when collected after sleep4, thus change in temperature may be a potential

stress or stimulus to activate tear-film PMNs. However, not all results support the activation

of tear-film PMNs during the 37oC incubation, as a decrease in CD66b was also observed

(activation associated with degranulation would have resulted in an increase in CD66b

expression). Since the downregulation of CD66b was also associated with lower levels of

CD16, this may also be indicative of apoptosis16. Large variations in the level of change in

expression between 0 and 4h incubation samples further suggested that tear-film PMNs may

not remain stable at 4hr. Similar observations have been made with blood leukocytes where

in vitro experiments tend to be limited to 2hr to preserve cell integrity33.

56

Our study also showed that centrifugation resulted in a downregulation of all our

selected cell membrane receptors except CD66b. Centrifugation is a necessary initial first

step to concentrate cells, but following centrifugation steps are often performed to wash cells,

re-concentrate cells, remove excess antibodies, etc. Since the centrifugation step was

performed before immunostaining, the reduced expression cannot be explained by the

antibodies being washed away. Internalization of surface receptors by tear-film PMNs due to

damage cell membrane integrity induced by exposure to shear force of the centrifugation

may be a possible explanation to the observed downregulation in receptor expression. This

would be corroborated by the fact that centrifugation of the T0 samples had a lesser impact

on the expression of surface receptors than in samples incubated for 4 hours: centrifugation

may induce some damage to cell integrity, which is further exacerbated in the 4hr sample

which may be undergoing activation and apoptosis (see above) due to the 4hr-incubation at

37oC. Interestingly, previous work with blood isolated leukocytes have either reported an

increase or no change following washing steps34,35. It is unclear why an upregulation in

CD66b (hence potential degranulation) is observed when all the other markers are being

downregulated.

A centrifugation step post staining (several centrifugation steps are often used post

staining to wash samples and remove unbound antibodies) resulted in a decrease in CD11b

expression and an increase in CD16, while CD45, CD55 and CD66b remained relatively

unchanged (data shown), further highlighting the potential impact of centrifugation. Not

only do our results emphasize the need to avoid extra centrifugation steps, it also highlights

the complexity in the underlying mechanisms of cell activation and membrane receptor up

and down regulation.

57

4.5. Conclusion

This study demonstrates that expression of membrane receptors on tear-film PMNs

exhibit different sensitivity to fixation, centrifugation and incubation and thus caution should

be exerted to ensure that experimental procedures will not affect marker of cell activation as

this has the potential to significantly impact result interpretation. Due to the drastic decrease

in expression of CD11b, CD16 and CD45, it is highly recommended to avoid exposing tear-

film PMNs to fixative prior to immunostaining. If samples need to be fixed, our results

suggest that post-fixation is preferred as post fixation induces minimal changes in activation

markers, except with CD16. Any additional centrifugation step after the initial

concentration/wash as well as long exposure to 37oC (4hrs or more) of tear-film PMNs

induce cell damage or activation and thus should be avoided unless appropriate controls are

in place. From the markers of activation assessed in this study, CD16, a marker of

degranulation, appeared to be most sensitive to experimental procedures and this may limit

its use in functionality study of tear film PMNs. The study allowed us to gain a better

understanding of how experimental procedures can affect the phenotype of tear film PMNs

which will help in the development of better protocols to characterize tear film PMNs and

assess their functionality in vitro.

58

Chapter 5

Comparison of Two Collection Methods and Investigation of the Response

of Tear-film Neutrophils to IL-8

5.1. Introduction

Neutrophils (also known as polymorphonuclear neutrophils or PMNs) represent

approximately 60% of the total leukocyte population and possess several efficient killing

mechanisms, such as phagocytosis and release of antimicrobial substances stored in granules,

to protect the host tissues from invading pathogens1.

It is known that a large number of PMNs can be collected from the ocular surface,

referred to as tear-film PMNs, after eyes are closed for a prolonged time2,3. Besides the

presence of tear-film PMNs, closed-eye tear fluid is rich in proteins, such as cytokines,

lysozyme, lactoferrin, secretory IgA and complement products, suggesting a state of sub-

clinical inflammation4. Interestingly, tear-film PMNs have been reported to be quiescent

upon stimulation and exhibit different expression of surface receptors associated with

activation on their cell membranes in comparison to blood-isolated PMNs2,5. This suggests

that tear-film PMNs may have already been primed and/or activated during their recruitment

to the ocular surface5,6. Although their origin is not established firmly, the hypothesis that

they originated from the conjunctival blood vessels and entered the ocular surface through

extravasation is supported by the low expression of L-selectin and high level of Mac-1

(CD11b)2. There are still many unknowns about their interaction with other immune cells,

their extensive presence in the nocturnal environment and their functionality, that need to be

further investigated.

59

To investigate and characterize their phenotypes, it is essential to have a collection

method that is non-invasive and able to consistently yield as many cells as possible for

subsequent in vitro experiments. Non-invasive methods used to collect cells from the ocular

surface have evolved over the years and have been used to harvest not only neutrophils but

also human corneal epithelial cells (hCECs). The “non-contact corneal irrigation chamber”

was used in 19867 to collect cells, but less than 200 hCECs and 8000 leukocytes were usually

obtained, preventing any cell phenotyping. The ocular surface cell collection apparatus

(OSCCA) developed by Peterson et al. allowed researchers to harvest more cells from the

ocular surface compared to previous methods8. While effective in cell collection, as it can

collect over hundreds of thousand neutrophils from the ocular surface9, the cost involved

with overnight studies limited the use of the OSCCA for collection of tear-film neutrophils,

as participants were required to sleep at the clinic. A new method, using a gentle eye wash,

was thus designed by Gorbet et al., which allowed participants to collect tear-film

neutrophils at home2. Each collection method has its drawbacks: there is an expensive

participant remuneration involved in OSCCA studies due to the required overnight stay,

whereas in the gentle eye method, inexperienced participants may lose cells due to the lack of

proficiency in collection procedures. To address this cost issue, we aimed to prevent the

overnight stay required by the OSCCA by evaluating the use of a patch to cover one of the

eyes (patch-OSCCA collection method) and asking the participants to come to the lab early

in the morning to perform cell collection using the OSCCA. The efficiency in collecting

cells was compared between the two collection methods (patch-OSCCA and gentle eye

wash) to determine which method would yield most cells, and if the phenotypes of tear-film

PMNs collected from the two collection methods differed from each other.

60

PMNs express various types of surface receptors, such as cytokine receptors, G-

protein coupled receptors, and Fc-receptors, which lead to different cell functional

responses10. Hence, evaluation of the expression of surface receptors provides information

on cell activation state. To characterize their immune response, tear-film PMNs are activated

in vitro with various stimuli. The stimuli used to characterize the response of tear-film

neutrophils have been phorbol-12-myristate-13-acetate (PMA; a potent synthetic chemical

activator of protein kinase C), lipopolysaccharides (LPS; a bacterial endotoxin), N-Formyl-L-

methionyl-L-leucyl-L-phenylalanine (fMLP; a chemotactic peptide), and calcium

ionophore2,5,6. However, these are not all physiological stimuli nor are they representative of

stimuli that tear-film neutrophils would typically encounter in the ocular environment.

Cytokines, such as tumor necrosis factor a (TNF-a)11, granulocyte-macrophage colony-

stimulating factor (GM-CSF)12 and interleukin-8 (IL-8)13 have been recognized to activate

blood-isolated neutrophils, however little is known on the response of tear-film neutrophils to

cytokines. Among these more physiological stimuli, IL-8 is a chemokine for neutrophils,

leading to activation and migration of blood leukocytes to infected sites through the actions

of L-selectin and Mac-114,15 and high levels of IL-8 have been observed in the closed-eye

environment16. Characterizing the response of tear-film neutrophils to IL-8 would provide

information on their activation state in the closed-eye environment as well as contribute

further evidence towards their phenotypic difference from blood leukocytes.

Our objectives were 1) to compare the number of cells collected between the patch-

OSCCA collection and the gentle eye wash method; and 2) to investigate the response of

tear-film PMNs to IL-8 and PMA, and compare it with that of blood-isolated PMNs. The

response to stimuli of tear film PMNs from the closed eye environment was evaluated

61

through the expression of surface receptors, CD11b (an adhesion and activation marker),

CD16 (a phagocytosis and degranulation marker), CD45 (a common leukocyte marker),

CD55 (a complement activation marker) and CD66b (a degranulation marker).


5.2.1. Reagents

Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies against human

CD11b and CD66b, R- phycoerythrin (PE)-conjugated monoclonal antibodies against human

CD55 and CD16 and R-phycoerythrin-cytochrome 5 (PE-Cy5)-conjugated monoclonal

antibody against CD45 were purchased from Becton Dickinson Pharmingen (San Diego, CA,

USA). Paraformaldehyde (PFA), IL-8 human, Trypan Blue, Histopaque-1077 and PMA were

from Sigma-Aldrich Co. (Oakville, Ontario, Canada). Phosphate-buffered saline (PBS) was

purchased from Lonza (Allendale, New Jersey, USA). Dulbecco’s modified eagle media

(DMEM) was purchased from Life Technologies (Burlington, Ontario, Canada). The eye

patch was ordered from Amazon Canada.

5.2.2. Study visits

The study received ethics clearance from the University of Waterloo Human

Research Ethics Committee. A total of 14 participants were recruited into the study, and

ocular health was verified through slit lamp observations, which were performed before and

between the collections. Participants were trained on the home collection procedure a day

before they actually performed it. Tear-film neutrophils were collected from each participant

five times over a one-month period: three collections at home (day 1, day 2 and day 29) and

two collections with the OSCCA took place in our laboratory (day 3 and day 30). The study

62

consisted of seven visits, including the two screening tests and five collections, as shown in

Figure 13.

Figure 13 - Schematic diagram of the study visits. ST: screening test; EW: the gentle eye wash method; PC: the patch-OSCCA collection method.

5.2.3. Gentle eye wash method

Participants used a previously established collection method described elsewhere 2,5.

To ensure they were comfortable with the collection procedure, training took place on day 0.

Briefly, 5 mL of sterile PBS was used to wash each eye, and the runoff was collected in a

sterile polypropylene tube. The sample was delivered to the lab within two hours of

collection. Cell suspension from each participant was centrifuged at 290xg for 10 min to

recover the cell pellet which was then resuspended in PBS. Cell count and viability were

determined under the microscope with Trypan Blue. Dilution to 100,000 cells/mL with PBS

was performed if applicable.

5.2.4. Patch-OSSCA Collection

Participants were instructed on wearing the patch the night before the collection day.

Patches were adjustable so as to firmly cover and fit the participant’s eye (figure 14b).

Participants were asked to cover one of their eyes with the provided patch just before going

to sleep, and then they slept at their homes as usual. Once they woke up in the morning, they

63

were required to come to the lab between 9 and 10 am. In order to prevent cell loss, they

were not allowed to take off the patches or wash their faces. Due to the potential associated

risks, participants were not allowed to drive with one eye covered. After the eye patch was

removed, tear-film PMNs were collected from the covered eye using the OSCCA (Figure

14a). Before the collection was performed, the funnel was rinsed with sterile distilled water,

and sterile PBS was warmed up to approximately 35 oC. Any part that was in direct contact

with the participant’s skin was wiped with ethanol and all other materials (tubing, needle,

etc) were autoclaved the day prior to collection. Once OSCCA preparation was complete,

participants were asked to place their head securely on a head-rest and face down. Next, they

were asked to hold their lower eyelid while the OSCCA operator held their upper eyelid. A

warmed and sterile PBS sprayed gently over the ocular surface via a pump for approximately

30 seconds. This saline solution washed cells from the ocular surface, and the eye wash was

collected into a sterile 15 ml polypropylene tube attached to the funnel. The suspension

samples were centrifuged at 290xg for 10 min to recover the cell pellet, which was then

resuspended in PBS. Cell count and viability were determined under the microscope with a

hemocytometer and Trypan Blue.

64

Figure 14 –OSCCA setup and the eye patch. a) the OSCCA; b) a patch with adjustable buckle; c) demonstration of wearing an eye patch.

5.2.5. Collection of blood-isolated PMNs

Peripheral blood was drawn from three medication-free and healthy participants and

added to a sterile polypropylene tube containing 5 U/mL of heparin (blood collection

happened on separate days for each participant). After centrifugation at 100xg for 10 min,

platelet-rich plasma was removed, and density-gradient centrifugation using Histopaque and

Polymorphprep (Axis Shield PoC AS, Oslo, Norway) was performed on the blood sample.

The isolated PMNs were washed three times with the first two washes in DMEM/10% FBS

with 5 mM EDTA (to prevent any leukocyte activation) and the last wash was performed in

sterile PBS. The purified blood-isolated PMNs were counted under the microscope with the

hemocytometer (Hausser Scientific, USA), and their viability was determined by Trypan

65

Blue. Dilution to 100,000 cells/mL was performed if applicable.

5.2.6. Cell stimulation and staining with fluorescent antibodies

Two stimuli were used in this study, PMA (2 µM, final concentration)2, a protein

kinase C (PKC) activator, and IL-8 (10 ng/mL, final concentration)17. Samples were divided

into three aliquots, which were unstimulated, PMA-stimulated, and IL-8 stimulated samples.

PMA stimulated samples were incubated for 20 min at room temperature2 while IL-8

stimulated samples were incubated for 30 min at 37oC17.

After the incubation with stimuli, 25µl of cell suspension was transferred into

corresponding labelled tubes containing antibodies against CD11b, CD16, CD45, CD55 and

CD66b, and tubes were incubated for 20 min in the dark at room temperature. Finally,

samples were diluted with DMEM/10% FBS and fixed with PFA (1%, final concentration).

All samples were analysed by flow cytometry after 24 h.

5.2.7. Flow cytometry

The CELLQuest Software (Becton Dickinson, Mountain View, CA, USA) on a

Becton Dickinson FACSCalibur flow cytometry (Mountain View, CA, USA) was used to

assess the expression of surface receptors on leukocyte samples. At least 2000 PMNs events

were acquired. Fluorescent values for each antibody (also known as Mean Fluorescent

Intensity, MFI) were recorded for all samples. To compare the changes in activation of tear-

film PMNs upon exposure to the various inflammatory stimuli, data is presented as the ratio

of the fluorescent intensities of stimulated samples versus unstimulated samples.

5.2.8. Statistics

All results are reported as means ± standard deviations. Statistical analysis was

66

performed using a paired sample t-test, Wilcoxon signed-rank test or an independent t-test to

evaluate if the difference of mean fluorescent intensities between unstimulated and

stimulated samples was significant. IBM SPSS Software (IBM Canada Ltd., Markham,

Ontario, CA) was used, and a p-value of less than 0.05 was required for statistical difference.

All graphs were plotted by GraphPad Prism Software (GraphPad Software, San Diego, CA,

USA).

5.3. Results

5.3.1. Patch-OSSCA collection versus home collection

As shown in Figure 15, the gentle eye wash method collected significantly more cells

than the patch-OSCCA collection method (p = 0.012), with average cell counts of

2.4 × 10A ± 1.6 × 10A versus 1.1 × 10C ± 1.6 × 10C, respectively.

Figure 15 - The total cells collected using the home collection method with the gentle eye wash (EW) and the patch-OSCCA collection method (PC). Cell numbers were counted under the microscope with the hemocytometer and Trypan Blue. n=6, individual participants.

67

The number of cells collected by the patch-OSCCA collection method was too low to

be analyzed via flow cytometry, so only cells obtained from the gentle eye wash method

were used to assess change in expression of surface receptors following exposure to

inflammatory stimulus. Due to the consistent low number of cells collected with the patch-

OSCCA collection, this method of collection was abandoned for the following 8 participants,

and the study visits were changed, as shown in Figure 16.

Figure 16 - Schematic diagram of the revised study visits. ST: screening test; EW: the gentle eye wash method.

The total number of cells collected using the gentle eye wash method among four

different days, day 1, 2, 29 and 30, were compared. As illustrated in Figure 17, there was a

significant difference between day 2 and 29 (p = 0.03), with the mean cell numbers of

3.1 × 10A ± 1.6 × 10A and 1.8 × 10A ± 1.4 × 10A, respectively. The difference observed

between the two days of collection is unlikely to be due to the lack of proficiency in

collection procedures, because participants were instructed and practiced on day 0, and they

did two collections before day 29. Furthermore, the volume of PBS they used to wash their

eyes was recorded when they brought the samples, and for all cell collections, less than 1 mL

of PBS was lost, indicating that participants performed the gentle eye wash method in an

appropriate manner.

68

Figure 17 - Total cell numbers collected using the gentle eye wash method over a one-month period. Cell numbers were counted under the microscope with the hemocytometer and Trypan Blue. For each collection day, n=14, values are presented from 14 participants.

5.3.2. Tear-film and blood-isolated PMNs responses to IL-8 and PMA in vitro

After stimulation with IL-8, there was no change in surface receptor expression in

tear-film PMNs as compared to unstimulated controls (p = 0.12), as shown in Figure 18a.

While there were some changes in level of expression with PMA-stimulated samples, these

did not reach statistical significance (p ≥ 0.1) except for CD16 (p = 0.003), as illustrated in

Figure 18b. This suggests that tear-film PMNs collected after sleep may be able to respond

mildly to PMA but not to IL-8.

69

Figure 18 - The expression of surface receptors of tear-film PMNs after sleep following stimulation with a) IL-8 and b) PMA. Tear-film PMNs were stimulated with IL-8 (10 ng/mL) or PMA (2 μM), and expression of surface receptors of CD11b, CD16, CD45, CD55 and CD66b was assessed through flow cytometry. Data is expressed as the ratio between stimulated (IL-8 or PMA) and unstimulated samples. The dotted line represents the resting (unstimulated) value. Results are presented as means ± standard deviations from 13 participants (n = 21 for PMA and n = 30 for IL-8). * Significantly different from unstimulated controls (p ≤ 0.005). On the other hand, in blood-isolated PMNs, IL-8 induced a drastic increase in the

expressions of CD11b, CD16 and CD45, while the levels of CD55 and CD66b remained

relatively unchanged (Figure 19a). Following exposure to PMA, as expected, significant

upregulation of CD11b and CD66b and downregulation of CD16 were observed, as shown in

Figure 19b. The large deviations observed in CD11b and CD66b are due to high responders,

a phenomenon often observed with PMA and these markers.

70

Figure 19 - The expression of surface receptors of blood-isolated PMNs following stimulation with a) IL-8 and b) PMA. Blood-isolated PMNs were stimulated with IL-8 (10 ng/mL) or PMA (2 μM), and expression of surface receptors of CD11b, CD16, CD45, CD55 and CD66b were assessed through flow cytometry. Data is expressed as the ratio between stimulated (IL-8 or PMA) and unstimulated samples. The dotted line represents the resting (unstimulated) value. Results are presented as means ± standard deviations from three participants (n = 3 for PMA and n = 4 for IL-8).

As shown in Figures 20 and 21, tear-film PMNs responded significantly differently

from blood-isolated PMNs to stimulation with IL-8 and PMA. IL-8 induced upregulation of

CD11b, CD16, CD45 and CD66b in blood-isolated PMNs was significantly higher than in

tear-film PMNs (p ≤ 0.032), while the upregulation in CD55 was not statistically significant

(p = 0.064) (Figure 20). Similarly, PMA induced significant upregulation in CD11b, CD45,

CD55 and CD66b in blood-isolated PMNs when compared to tear film PMNs (p ≤ 0.001).

PMA induced a similar downregulation in CD16 expression in both tear film and blood

isolated PMNs. All in all, these results suggest that both IL-8 and PMA failed to activate

71

tear-film PMNs as compared to blood-isolated PMNs, further confirming previous results on

the difference in phenotypes between blood and tear film PMNs.

Figure 20 - The expressions of surface receptors of blood-isolated PMNs and tear-film PMNs following stimulation with IL-8. PMNs were stimulated with IL-8 (10 ng/mL), and their expression of surface receptor of CD11b, CD16, CD45, CD55 and CD66b was assessed through flow cytometry. Data is expressed as the ratio between stimulated (IL-8) and unstimulated samples. The dotted line represents the unstimulated value. Results are presented as means ± standard deviations from 13 participants collecting tear-film PMNs several times (n = 30) and 3 participants donating blood at least once (n=4). * Significantly different from blood-isolated PMNs (p < 0.03).

72

Figure 21 - The expression of surface receptors of blood-isolated PMNs and tear-film PMNs following stimulation with PMA. PMNs were stimulated with IL-8 (10 ng/mL), and their expression of surface receptor of CD11b, CD16, CD45, CD55 and CD66b was assessed through flow cytometry. Data is expressed as the ratio between stimulated (PMA) and unstimulated samples. The dotted line represents the unstimulated value. Results are presented as means ± standard deviations from 13 participants collecting tear-film PMNs several times (n = 21) and 3 participants donating blood for PMNs isolation (n = 3). * Significantly different from blood-isolated PMNs (p ≤ 0.001). To compare the phenotype of unstimulated (resting) tear-film and blood-isolated

PMNs, the level of expression of the membrane receptors are presented as raw fluorescent

values (also referred to as mean fluorescent intensities (MFI)) in Table 5. Note that all data

was collected on the flow cytometer using the same voltage settings and thus any difference

in fluorescence is due to a difference in level of expression on the cell membrane. As shown

by the MFI values, the level of expression of CD11b, CD55 and CD66b on unstimulated

73

tear-film PMNs were significantly higher than that of blood-isolated PMNs, and CD16

expression was significantly lower in tear-film PMNs (p < 0.001). It is also important to

highlight the high standard deviation observed in the expression of CD16 for tear film PMNs:

with some participant’s PMNs exhibiting the lowest fluorescent intensity of 3 while others

have as high as 120 at the time of collection, indicating a significant range of CD16

expression across participants. We were unable to organize the participants into groups

based on their CD16 fluorescent intensities of CD16, as the level of expression of CD16

changed between the various days of collection for the same participant.

Table 5 - Expression of selected membrane receptor on unstimulated tear-film and blood-isolated PMNs. Values are reported as mean fluorescent intensities (MFI).

CD markers Tear-film PMNs Blood-isolated PMNs

CD11b 358 ± 79* 48 ± 22

CD16 28 ± 31* 841 ± 198

CD45 71 ±14* 47 ± 17

CD55 154 ± 44* 55 ± 26

CD66b 297 ± 83* 35 ± 22

Results are presented as means ± SD from 13 participants collecting tear-film PMNs several times (n = 53) and 3 participants donating the blood-isolated PMNs (n = 6). * Significantly different from blood-isolated PMNs (p < 0.001).

74

5.3.3. Comparing the phenotype of tear-film PMNs between collection days for each

participant

There was a two-week gap between repeated cell collections which divided the four

cell collections into two parts: the first two took place on day 1 and 2, and the other two were

performed on day 29 and 30. As there were not enough tear-film PMNs obtained on day 30

for most of the participants, only cells collected on day 1, 2 and 29 were used to compare

their phenotypes to each other. As shown in Figure 22, there was no significant difference

detected in the stimulation ratio for surface receptors of IL-8-stimulated tear-film PMNs

among the three days (p < 0.15) except for CD55 where its expression on day 1 was

significantly higher than that of day 29 (p = 0.04) with the mean ratio of 1.10 and 0.96,

respectively. On the other hand, when tear film PMNs were exposed to PMA, only the

expression of CD16 on day 2 was significantly lower than that on day 29 (p = 0.01) with a

ratio of 0.39 and 0.55, respectively.

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Figure 22 – Change in IL-8 and PMA stimulation of tear-film PMNs collected at the beginning and end of the month. a) CD11b; b) CD16; c) CD45; d) CD55; e) CD66b.

76

Figure 23 reports the level of expression of membrane receptors on unstimulated tear-

film PMNs at day 1, 2 and 29. Except for CD55 and CD16, the level of expression of membrane

receptors remained constant across the collection days. The expression of CD55 on day 1 was

significantly higher than that on day 2, with a mean MFI of 176 and 143 (about 81% reduction

on day2) (p = 0.006). Although the changes in level of expression of CD16 across the days did

not reach statistical significance, as can be seen in Figure 11b, a spread in CD16 expression was

observed not only across days but within single participant collection data, whereby on day 1, a

MFI of 15 would be recorded and then 58 on day 2 or day 29.

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Figure 23 – Change in the level of unstimulated expression of membrane receptors of tear-film PMNs collected at the beginning and end of the month. A) CD11b; b) CD16; c) CD45; d) CD55; e) CD66b.

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5.4. Discussion

An ideal non-invasive collection method should collect maximal cells without

causing any artefactual changes. The gentle eye wash method was successfully designed to

collect leukocytes from the ocular surface, and the total cells obtained have been reported to

be significantly higher than the method using the non-contact corneal irrigation chamber2,3.

However, the number of cells collected may be dependent on the participant’s proficiency in

collection procedures. Hence, a training session before the actual collection is usually

recommended. In addition, large variations among expressions of surface receptors have

been observed not only in our own results but also of others2,5, which may be due to

individual difference in the response or may also be due to the isolation or preparation

procedures. The latter was addressed in the previous chapter. Thus, in this study, we aimed

to determine which method could yield the most cells and also provide more reliable results.

The gentle eye wash method collected 200 times more cells than the patch-OSCCA

collection, indicating that the gentle eye wash method is the most effective method to collect

tear-film PMNs at home so far. Unlike the gentle eye wash method that collects cells

immediately upon awaking, the patch-OSCCA collection takes a much longer time,

approximately 40-60min, as participants need to come to the lab to have the OSCCA cell

collection procedure performed on their eye. Hence, during the time between home and the

lab, participants will likely slightly open their eye unintentionally under the patch, resulting

in significant cell loss.

While the gentle eye wash collected more cells, from our study, it appears that the

number of cells collected with the gentle eye wash is not consistent on different days. Cell

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counts were higher on day 2 compared to day 1. This may be because participants practiced

their collection skill in day 1 and were thus more familiar on day 2. However, we cannot

explain why there is a significant decrease in collection from day 2 to day 29 and a further

decrease from day 29 to day 30. This reduction is unlikely due to the collection procedure,

as by then the participants are familiar with the collection methods and the volume of cell

suspension during cell collection remained consistent across all collection days. Weather

(cold versus warm, etc) or other health related matters could also not account for the

decrease/change observed. The possible factors contributing to poor collection may be more

psychological conditions, whereby around the last collection days, the participants may be

more careless in following the protocol of collecting immediately on awakening thinking that

the study is coming to an end.

While differences were observed in the total number of cells collected among the

various days for each participant, limited changes occurred in the level of expression of the

membrane receptors on the collected cells, except for CD55 and CD16. In unstimulated tear-

film PMNs, CD55 showed an 25% reduction on day 2 as compared to day 1. The different

level of expression of CD55 between days may suggest that complement activation at the

ocular surface may be highly variable. The changes observed in CD16 expression may be

due to underlying closed-eye conditions, as a downregulation is correlated with elastase

release. It remains unclear why CD16 and CD55 expression remains so variable, but the

variability observed in our results with CD16 and CD55 expression would suggest that these

membrane receptors may not be reliable indicator for future diagnostic and phenotyping

purposes. Collectively, the intra difference (within a single subject) expression of CD11b,

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CD45 and CD66b was relatively small, suggesting that these markers can be used more

reliably and that only one cell collection would be necessary to identify cell phenotype.

IL-8 acts as a potent chemoattractant for PMNs and is able to induce various

functions, such as regulating the transmigration from the vessels to the infected sites,

respiratory burst, exocytosis and phagocytosis18. IL-8 has been shown to cause an increase in

the expression of Mac-1 (CD11b/CD18) to promote adhesion of PMNs14,15. In our study, as

expected, IL-8 induced cell activation in blood-isolated PMNs, as identified by CD11b

upregulation. However, no change was observed in tear-film PMNs upon stimulation with

IL-8. Almost 95% of Mac-1 is stored in cytoplasmic granules and can be rapidly mobilized

to the PMNs cell surface during/after activation1,19. Hence, the impaired upregulation of

CD11b of tear-film PMNs may be caused by the shortage of cytoplasmic stores, as tear-film

PMNs already show a high level of CD11b expression (see Table 5). Our results suggest that

tear-film PMNs may already be in an activated state and unable to respond further.

Both CD55 and CD66b levels on blood-isolated and tear-film PMNs remained similar

to unstimulated samples, suggesting that IL-8 stimulation may have no impact on their

expression. Research has shown that CD66b is less sensitive to cytokines compared to

CD11b and CD62L20. In addition, the upregulation of CD55, which is known as decay-

accelerating factor (DAF), is dose-dependent19; no increase in CD55 expression was

observed with an IL-8 concentration of 4 ng/mL, whereas a significant upregulation of CD55

was detected with 400 ng/mL19. Hence, the lack of upregulation in CD66b and CD55 in this

study may be due to the insensitivity to IL-8 and the low concentration of IL-8 used

(10ng/ml), respectively.

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Gorbet et al. showed that tear-film PMNs were unable to respond to fMLP2. The

fMLP and IL-8 receptors (IL-8Rs) are members of the seven-transmembrane spanning

receptor (STMR) coupled to a G protein21, so IL-8 and fMLP may have some similarities in

the signal transduction pathways22. Wu et al. demonstrated that the binding of IL-8 to G-

coupled protein receptor leads to the dissociation of the subunits, which activates the

phospholipase C (PLC) and eventually increases intracellular calcium ions22. The

involvement of PLC, ras/raf, mitogen activated protein kinase (MAPK), and

phosphoinositide 3-kinase (PI3K) contribute to the IL-8 stimulated pathways21, which are

also involved in the fMLP-stimulated pathway23. Since their signal transduction pathways

involve similar components, the inability for tear-film PMNs to respond to IL-8 may occur

because of the impairment of compartments within the pathways shared with fMLP or/and

the receptors.

Another possible factor contributing to the inability of tear-film PMNs to upregulate

their surface receptors upon IL-8 stimulation may also be due to a lack of functionality in G-

protein coupled-receptors.

Apart from the signal transduction pathway, downregulation of IL-8Rs may also

cause the lack of response to IL-8 of tear-film PMNs24. Pignatti et al. showed that the

numbers of IL-8Rs expressed on PMNs isolated from sputum (a “fluid” induced from the

central airways25) collected from non-severe asthma patients were lower than on blood PMNs

from healthy people24. They suggested that one of the possible causes was that exposure to

IL-8 was inducing the internalization of IL-8Rs24. Studies have already demonstrated that

the concentration of IL-8 increases during sleep6,26. Hence, the inability to elicit an

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activation response from tear film PMNs may be due to the internalization of IL-8Rs when

exposed to IL-8 in the closed-eye environment. More work is required to confirm such

hypothesis as the expression of IL-8Rs on tear-film PMNs has not been investigated so far.

Furthermore, as discussed above, the activation mechanisms for PMNs are complex, so

further experiments will be required to understand the underlying mechanisms to fully

explain the lack of response upon IL-8 stimulation.

Upon PMA stimulation, tear-film PMNs showed small magnitudes of changes in the

expression of surface receptors in comparison to blood-isolated PMNs, except for CD16.

The downregulation of CD16 upon activation has been shown to be associated with the

cleavage by proteases, specifically neutrophil elastase5,27. Both tear-film and blood-isolated

PMNs showed significant downregulation upon PMA-stimulation suggesting that release of

primary granules containing elastase which can shed off the CD16 could occur in tear film

PMNs upon PMA stimulation. It is important to note that the large variations observed in the

ratio of CD16, especially in PMA-stimulated tear-film PMNs, was due to the fact that in

some participants the unstimulated samples already had minimal CD16 expression and thus a

further decrease below background was not possible. However, all in all, our results showed

that CD16 was downregulated on tear-film PMNs stimulated by PMA, suggesting that some

degranulation occurred in these cells upon exposure to PMA.

CD55 is present in secretory vesicles19, which is one of the four storage granules in

PMNs and has been shown to be quickly mobilized to the cell membranes after stimulation28.

Therefore, the upregulation of CD55 is due to the release of secretory vesicles, which can be

seen in both tear-film and blood-isolated PMNs after exposure to PMA. However, the

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magnitude of CD55 upregulation is much lower in tear-film PMNs, suggesting that most of

the secretory vesicles may have already been released by activated tear-film PMNs in the

closed-eye environment5. PMA mimics the action of diacylglycerol (DAG) and directly

binds and activates the protein kinase C (PKC). It has been shown that CD18 is

phosphorylated during PKC activation29. Therefore, the mechanism of PMA upregulation of

Mac-1 (CD11b/CD18) may occur via the activation of PKC. The lesser upregulation of

CD55 with tear film PMNs correlates well with the lower magnitude of Mac-1 upregulation

in tear-film PMNs as this can likely be explained by prior granules and secretory vesicle

release in the closed eye environment.

As shown in Table 5, the level of expression of CD11b, CD55, and CD66b in resting

tear-film PMNs were more than doubled that of unstimulated blood-isolated PMNs, while for

CD16, it was approximately 20 times lower. All these markers are cell activation markers

and an up or down regulation is indicative of an activated phenotype, and thus these results

further contribute to the hypothesis that tear-film PMNs may have already been activated in

the closed-eye environment. PMNs found in the mouth30, placenta31, and the lungs32 show a

similar pattern of expression of surface receptors.

5.5. Conclusion

In conclusion, the patch-OSCCA collection is not effective in collecting cells in

comparison with the gentle eye wash method. While a high number of cells were collected

with the gentle eye wash method, significant changes in the total number of cells collected

were observed among the various days of cell collection for each participant. However, the

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level of expression of cell activation membrane receptors on the collected cells remains

relatively unchanged for each participant, except for CD16 and CD55 (whose expression is

associated with the release of granules and vesicles). While variations in CD16 and CD55

expression might be an indication of what may have happened in the ocular environment

during sleep, the significant changes that occur within a participant and across the population

may make it difficult to use CD16 and CD55 for diagnostic purposes. Our results, however,

suggest that one cell collection from a participant provides a reliable means of assessing the

tear film PMNs phenotype with another marker of cell activation.

Our study also demonstrated that tear film PMNs collected from the closed eye

environment were unable to up or down regulate cell activation membrane receptors upon

IL-8 stimulation, suggesting an impairment that may be explained by prior exposure to IL-8

and activation in the closed-eye environment. However, the small response to PMA-

stimulation was also observed suggesting that other potential mechanisms may be involved to

lead to a quiescent state in tear film PMNs. Further investigations will be required to gain a

better understanding on the phenotype and state of activation of tear film PMNs.

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Chapter 6

Characterization of Respiratory Burst of Tear-film Neutrophils by

Luminol-Enhanced Chemiluminescence

6.1. Introduction

Neutrophils (PMNs) comprise approximately half of the total circulating white blood

cells, which are the hallmark of multi-lobed nuclei and cytoplasmic granules1. PMNs are one

of the population of innate immune cells playing an essential role in protecting the host

tissues against foreign particles, evoking multiple functional responses upon activation by

stimuli. This includes the generation of reactive oxygen species (ROS), the release of

antimicrobial substances from granules, and phagocytosis. PMNs, like other immune cells,

are heterogeneous, meaning they exhibit distinct phenotypes in different tissues under normal

physiological conditions. The eye represents a unique physiological environment due to its

immune privilege2, thus recruitment of immune cells to the ocular surface is restricted.

PMNs isolated from the ocular surface exhibit an interesting nocturnal-diurnal pattern, as

while few PMNs can be found on the ocular surface during the day in healthy eyes, hundreds

of thousands of PMNs can be detected after eye closure for a prolonged time3. The

underlying regulatory mechanism leading to this pattern has not yet been elucidated,

although chemotactic molecules (IL-8 and complement products) are believed to play a role4.

Two research groups have investigated the state of activation of tear film PMNs upon

collection from the eyes after sleep by evaluating the expression of cell activation membrane

receptors3,5. Unlike blood PMNs, tear-film PMNs expressed high levels of CD66b (a

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degranulation marker) and CD11b (a cell adhesion and activation marker) but low levels of

CD62L (L-selectin)3,5, suggesting that they probably have already been activated on the

ocular surface upon arrival through the extravasation or due to their prolonged presence in

the closed eye environment5. A recent study has suggested that tear-film PMNs may have an

impaired capability of phagocytosis6 and are also unable to upregulate or downregulate cell

activation membrane receptors in response to various inflammatory stimuli3,5.

Respiratory burst, as one of their essential killing mechanisms, has been extensively

investigated in blood PMNs. ROS generated during the respiratory burst can not only

eliminate pathogens but can also cause severe damage to surrounding tissue if over-

produced. An increase in oxygen consumption by PMNs has been detected during

phagocytosis, as the activated nicotinamide adenine dinucleotide phosphate oxidase

(NADPH oxidase) reduces the molecular oxygen to the superoxide anion (O"#)1,7. PMNs

isolated from patients suffering from chronic granulomatous disease (CGD) can engulf

microorganisms but cannot generate ROS, resulting in a failure in killing invading

microorganism8. This suggests that the respiratory burst is crucial for PMNs to perform their

functions. NADPH oxidases can be found in both PMNs plasma membrane, which

contribute to the extracellular superoxide anions, and granule membranes, that release

superoxide anions in an intracellular manner9. The superoxide anion is spontaneously

converted to hydrogen peroxide and reacts with a chloride ion to form hypochlorous acid, a

potent antimicrobial agent, under the catalyzation of myeloperoxidase1. The most efficient

stimuli used to trigger the respiratory burst in PMNs are opsonized microorganisms,

complement fragment C5a, N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP),

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platelets activating factor (PAF), and phorbol-12-myristate-13-acetate (PMA)1. The

interaction between these stimuli and their corresponding receptors activates a series of

intracellular signalling pathways to induce different PMNs functional responses (except

PMA, which acts directly on protein kinase C (PKC)).

Detection of ROS can be achieved by multiple methods, such as fluorescent and

chemiluminescent probes, spectrophotometry, and chromatography10. Among these

methods, luminol-enhanced chemiluminescence is easy to operate and also provides a kinetic

measurement of ROS formation. Although the superoxide anion is able to spontaneously

dismutate to hydrogen peroxide, hydrogen peroxide is relatively stable and cannot react with

luminol to emit light11. Hence, this assay is efficient to detect superoxide anions from both

intra- and extracellular compartments, as luminol is cell membrane permeable10,12.

Although tear-film PMNs are shown to be non-inflammatory upon stimulation, it is

worthwhile to investigate their ability to generate ROS and to compare this process with

blood-isolated PMNs to determine if the two subsets of PMNs show similar patterns of the

respiratory burst. Also, while Gorbet’s research group has previously assessed the level of

ROS produced by tear-film PMNs through flow cytometry, it can only provide endpoint

values and cannot assess extra-cellular ROS generation. In this study, luminol-enhanced

chemiluminescence was performed to characterize the kinetics of ROS production in tear-

film PMNs. The stimuli selected to activate tear-film PMNs were fMLP, a chemotactic

peptide secreted by bacteria, PMA, a chemical stimulus that can directly activate PKC, and

lipopolysaccharides (LPS), fragments from the outer membrane of gram-negative bacteria.

We hypothesized that tear-film PMNs generation of ROS in response to the selected stimuli

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would be lower than that of blood-isolated PMNs.


6.2.1. Materials

Endotoxin-free reagents, medium and sterile tubes were used in this experiment to

avoid undesired activation of PMNs. LPS (Escherichia coli serotype 0111:B4), PMA, fMLP,

Hanks's balanced salt solution (HBSS) and luminol (5-amino-2,3-dihydroxy-1,4-

phthalazinedione) were ordered from Sigma-Aldrich Co. (Oakville, Ontario, Canada).

Phosphate-buffered saline (PBS) was purchased from Lonza (Allendale, New Jersey, USA).

Dulbecco’s modified eagle media (DMEM) was purchased from Life Technologies

(Burlington, Ontario, Canada).

6.2.2. Blood-isolated PMNs isolation:

This study received ethics clearance from the University of Waterloo Human

Research Ethics Committee. PMNs were isolated from fresh blood drawn from four healthy

and asymptomatic participants, who did not take any anti-inflammatory medication for at

least 72 hours. Around 10 ml of blood was collected from each participant and immediately

added to a tube containing 5U/ml of heparin. Blood was then centrifuged at 100xg for 10

min, and platelet-rich plasma was removed. DMEM/10% FBS was added to dilute the blood

in a ratio of 1:1 and 5 mM EDTA was added. Diluted blood was then transferred to the tube

containing PolymorphPrep (Axis Shield PoC AS, Oslo, Norway) and Histopaque (Oakville,

Ontario, Canada), resulting in a 1:1 ratio of diluted blood and the two density gradient media,

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followed by centrifugation at 930xg for 25 min. PMNs (located in the bottom band) were

carefully transferred into a new 15 mL polypropylene tube followed by three washes (at

280xg for 10 min) with the first two washes in DMEM/10% FBS with 5 mM EDTA and the

last one in PBS. PMNs were resuspended in PBS, and cell count was performed.

6.2.3. Tear-film PMNs collection

Tear-film PMNs were collected after sleep from nine non-lens wearers without any

ocular disease or complications, as described in a previous study3. Briefly, participants were

trained to collect their cells via the gentle eye wash method, using sterile PBS to wash the

ocular surface, and the run-off was collected in a sterile polypropylene tube. Samples were

brought to the lab and the cell suspension was centrifuged at 290xg for 10 min, followed by

the resuspension in PBS. Cell count and cell viability were assessed using the

hemocytometer with Trypan Blue.

6.2.4. Luminol-enhanced chemiluminescence

Chemiluminescence (CL) measurements were evaluated on the Cytation 5 Cell

Imaging Multi-Mode Reader (BioTek) using 96-Well Cell Culture White Microplates

(Greiner bio-one, Frickenhausen, Germany). Each well was set to test one stimulus, and each

had a total reaction volume of 200 $L containing 105 PMNs, 1 mM luminol, its

corresponding stimulus (2 $M PMA, 1.5 $M fMLP and 2 $g/mL LPS) 3 and hanks's

balanced salt solution (HBSS; components are listed in Table 6) which was added to 200µL.

The controls were performed in each assay and contained the same concentration of reagents

but no stimulus (ie HBSS was added to complete volume to 2200µL). To obtain kinetic

90

curves, CL emissions were recorded at 37 oC for 120 minutes at 3-minute intervals (as show

in Figure 24).

Figure 24 – Examples of chemiluminescence kinetic response recorded with tear film PMNs when exposed to a) PMA, b) fMLP and c) LPS. Values are recorded as CLU, chemiluminescent unit.

Table 6 – Components of HBSS 13. Components g/L

Calcium Chloride (anhydrous) 0.1396

Magnesium Sulfate (anhydrous) 0.09767

Potassium Chloride 0.4

Potassium Phosphate Monobasic (anhydrous) 0.06

Sodium Chloride 8.0

Sodium Phosphate Dibasic (anhydrous) 0.04788

D-Glucose 1.0

6.2.5. Statistics

After careful analysis of the CL kinetic graphs, the CL response at different time

points was selected for statistical analysis, 0, 3, 6, 12, 24, 48, 60 min for fMLP, 0, 9, 27, 54,

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60 min for PMA, and 0, 12, 21, 33, 45, 60 min for LPS. Changes in CL was expressed as the

ratio of stimulated CL emissions to control CL emissions (CLstimulated/ CLcontrol), which were

then plotted over time. Results are reported as means ± standard deviations, except when the

representative CL curves are presented. Kinetic curves and area under the curve (AUC) were

computed by GraphPad Prism Software (GraphPad Software, San Diego, CA, USA). In

order to assess the statistical significance, an independent t-test or Mann-Whitney test was

performed using IBM SPSS Software (IBM Canada Ltd., Markham, Ontario, CA), and a p-

value less than 0.05 was required for statistical difference.

6.3. Results

6.3.1. ROS production in fMLP-stimulated tear-film and blood-isolated PMNs

Respiratory burst was initiated by the addition of fMLP, and the generation of ROS

was measured by chemiluminescence emissions. Changes in kinetics in ROS production of

fMLP-stimulated tear-film and blood-isolated PMNs over 60 min are reported in Figure 25.

Except at 0, 3 and 6 min, ROS production, as identified by emitted CL, by fMLP-stimulated

tear-film PMNs, was close to background level, at 12-, 24-, 48- and 60-minute time points

and was significantly lower than blood-isolated PMNs (p ≤ 0.007). Tear-film PMNs ROS

production showed a transient and sharp increase around the 3-minute time point, which was

higher than blood-isolated PMNs but did not reach statistical significance (p = 0.07). Blood-

isolated PMNs CL exhibited two peaks at 3 and 24 minutes, whereas tear-film PMNs only

showed one peak at 3 minute and then continuously declined to the baseline level. This high

generation of ROS within 3 minute after exposure to fMLP has been previously observed by

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Zhu et al11.

Figure 25 - Changes in chemiluminescence of tear-film and blood-isolated PMNs when exposed to fMLP. Results are reported as ratio of fMLP-stimulated CL emissions over control (unstimulated) sample CL emissions. Neutrophils (105 cells/well) were transferred to the well containing 1 mM luminol, 1.5 'M fMLP and HBSS. Generation of ROS was kinetically measured using the Cytation 5 at 37oC, as described under Materials and Methods. Values are presented as means ± standard deviations from nine healthy participants collecting tear-film PMNs several times (n = 10) and four participants donating their blood for isolation of PMNs (n = 4). * Significantly different from unstimulated controls (p≤ ). ))+).

6.3.2. ROS production of LPS-stimulated tear-film and blood-isolated PMNs

The response to LPS was also investigated and is reported in Figure 26. LPS could

not induce a respiratory burst in tear-film PMNs, as shown by the CL ratio being close to 1,

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indicating no difference in CL emissions between the stimulated and unstimulated samples.

On the other hand, blood-isolated PMNs started to continuously produce ROS at 12 min.

Starting at around 30 min of stimulation, production of ROS was significantly different

between blood-isolated and tear-film PMNs (p ≤ 0.02)

Figure 26 - Changes in chemiluminescence of tear-film and blood-isolated PMNs when exposed to LPS. Results are reported as ratio of LPS-stimulated CL emissions over control (unstimulated) CL emissions. Neutrophils (105 cells/well) were transferred to the well containing 1 mM luminol, 2 µg/mL of LPS and HBSS. Generation of ROS was kinetically measured using the Cytation 5 at 37oC, as described under Materials and Methods. Values are presented as means ± standard deviations. n = 6, tear film PMNs from different participants and n=4 for blood isolated PMNs from different blood donors * Significantly different from unstimulated controls (p ≤ 0.02).

94

6.3.3. Production of ROS in PMA-stimulated tear-film PMNs and blood-isolated PMNs

To determine the responses of tear-film PMNs to other stimuli, PMA was selected, as

a synthetic PKC activator that can directly bind to and activate PKC 1, whereas fMLP and

LPS are receptor-dependent stimuli. Upon exposure to PMA, both tear-film and blood-

isolated PMNs CL showed a peak of ROS generation and exhibited similar trend although

the magnitudes were different, as shown in Figure 27. Blood-isolated PMNs CL had a sharp

increase in ROS production at 9 min, and then CL continuously declined. On the other hand,

tear-film PMNs CL reached their highest production level at 27 min, indicating a much

slower ability to generate ROS upon PMA stimulation when compared to blood-isolated

PMNs. Similar to blood-isolated PMNs, CL values gradually decreased afterwards. There

was no significant difference between ROS production of tear-film and blood-isolated PMNs

at all time points (p ≥ 0.22) except at 0 min, which is further discussed in section 6.3.5.

Comparing the PMA-stimulated CL to fMLP- and LPS-stimulated CL, both tear-film and

blood-isolated PMNs showed a higher and prolonged production of ROS.

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Figure 27 – Changes in chemiluminescence of tear-film and blood-isolated PMNs when exposed to PMA. Results are reported as ratio of PMA-stimulated CL emissions over control CL emissions. Neutrophils (105 cells/well) were transferred to the well containing 1 mM luminol, 2 µM PMA and HBSS. Generation of ROS was kinetically measured using the Cytation 5 at 37oC, as described under Materials and Methods. Values are presented as means ± standard deviations from eight healthy participants collecting tear-film PMNs several times (n = 10) and three participants donating their blood for isolation of PMNs (n = 3). * Significantly different from unstimulated controls (p = 0.02).

As seen in Figure 27, large standard deviations in ROS production of tear-film PMNs

were present due to differences in individual responses. Two distinct groups of response

could be observed, with some participants presenting greater changes in CL than others: two

groups could be identified based on the magnitude of the ratio. Participants who had a ratio

above 10 were classified as high responders (suggesting a high potential in producing ROS)

while the others, with a ratio below 10, were classified as low responders. Individual results

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are summarized in Table 7. An independent t-test confirmed that the means between the two

groups of participants were significantly different (p < 0.001), further emphasizing how

distinct these two groups were. It is interesting to note that the HR group was composed of

female participants in their early twenties while the LR group consisted of both female and

male in their late twenties, suggesting tear-film PMNs isolated from younger female

participants may tend to be hyperactive. However, given our small sample size (n=8), this

needs to be further examined.

As shown in Figure 28, once groupings were used, standard deviations evidently

decreased, but most importantly the two distinct responses (LR and HR) in participants could

be clearly observed.

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Table 7 - Summary table of ROS generation potentials for high responders and low responders.

Participants

Arbitrary ID#

Age Sex Highest ratio of

CLstimulated/CLcontrol

High responders *

#1 25 F 17.12

#2 24 F 16.19

#3 24 F 19.68

#4 21 F 14.86

Mean 16.97

SEM 2.04

Low responders

#5 29 F 5.79

#6 22 M 5.91

#7 32 F 4.38

#8 23 M 6.76

Mean 5.71

SEM 2.69

* significantly different from low responders, p < 0.001

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Figure 28 - Changes in CL of PMA-stimulated tear-film PMNs of high responders (HR) and low responders (LR). Mean and standard deviation were computed for each grouping at each time point (t = 0, 9, 27, 54, and 60 min) (n = 10, from 4 different participants in each group). * Significantly different from LR (p ≤ 0.02).

6.3.4. Comparison of area under the curve and endpoints between tear-film and blood-

isolated PMNs

The above kinetic CL curves (Figures 2 to 5) showed the real-time ROS production

by tear-film and blood-isolated PMNs over 60 minutes. From such curves, the area under the

curve (AUC) can be computed to reflect the quantity of ROS released from tear-film and

blood-isolated PMNs after exposure to different stimuli, as shown in Figure 29a. The overall

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ROS generation for blood-isolated PMNs were higher than that of tear-film PMNs; with

AUCs for fMLP-stimulated (p = 0.003) and LPS-stimulated (p = 0.031) blood-isolated PMNs

CL being significantly higher than tear-film PMNs CL. However, AUCs of PMA-stimulated

tear-film and blood-isolated PMNs were not significantly different (p = 0.184). Since two

responder groups were observed with PMA-stimulation, we also compared the AUC between

the blood-isolated and tear-film PMNs based on the HR and LR groupings. Interestingly,

there was no significant difference in the mean AUC between HR of blood-isolated and tear-

film PMNs (p = 0.17), whereas the mean AUC between LR of blood-isolated and tear-film

PMNs, 147 and 271, respectively, were significantly different (p = 0.008).

The chemiluminescence assay was run for 120 minutes; the CL values at the

endpoints were recorded and computed as ratio as shown in Figure 29b. Changes in tear-film

PMNs CLs stimulated by fMLP, LPS and PMA were significantly lower than in blood-

isolated PMNs CLs at 120 minutes (p ≤0.03).

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Figure 29 – Comparison of area under the curve (AUC) and the ratio of changes at the endpoint (t = 120 min) between tear-film and blood-isolated PMNs for all three stimuli. A) AUC computed by GraphPad Prism Software; B) changes in CL at 120 min. Values are presented as means ± standard deviations. * Significantly different from blood-isolated PMNs (p < 0.05).

6.3.5. CL values at time zero in unstimulated samples.

To determine if tear film PMNs collected from the closed eye environment may be in

an activated state, the level of spontaneous ROS production in the unstimulated samples was

compared to that of blood-isolated PMNs at early time points (0 and 3 minutes). As shown

in Table 8, CL values of unstimulated tear film PMNs was significantly higher compared to

blood-isolated PMNs, suggesting that tear film PMNs collected after sleep were in a state of

activation and had been exposed to a pro-inflammatory environment.

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Table 8 - ROS production in unstimulated tear film and blood-isolated PMNs Time point Tear film PMNs (CLU) Blood-isolated PMNs (CLU)

0 min 38 ± 12* 20 ± 8

3 min 46 ± 20* 15 ± 6

CLU: chemiluminescence unit. Mean ± SD, n= 15, for tear film PMNs from 8 different participants and n=6, for blood-isolated PMNs from 4 different blood donors. *significantly different from blood isolated PMNs, p < 0.01

6.3.6. Participant with seasonal allergy

One participant with seasonal allergy was also unintentionally recruited during the

study. Tear-film PMNs were collected and their ability to generate ROS after stimulation

was assessed, as shown in Figure 30. The data of this participant was excluded from the

above results but is presented as an interesting case study (n = 1), to provide some

information on the potential effects of ocular allergy on ROS production in tear film PMNs.

The overall tear-film PMNs CLs of the allergy participant was more than two times higher in

comparison to healthy participants, indicating that the collected tear film PMNs were in a

highly reactive and pro-inflammatory state. Apart from the fact that higher levels of

inflammatory cytokines 14 have been observed in closed-eye tears of allergic participants,

there is currently limited knowledge on how ocular allergy affects the leukocyte population

and its phenotype. While this is but one participant, these preliminary results highlight how

an inflammatory condition affects ROS production and how research in that area could

contribute significant knowledge.

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Figure 30 – Kinetic changes in CL of tear-film PMNs collected from the allergy participant over 60 min compared to asymptomatic/healthy participants. Results are reported as the ratio of stimulated CL emissions over control CL emissions, and each colored line represents one participant. The purple line identifies the ocular allergy symptomatic participant. A) fMLP-stimulated tear-film PMNs; B) LPS-stimulated tear-film PMNs; C) PMA-stimulated tear-film PMNs.

6.4. Discussion

Luminol reacts with ROS and releases energy in the form of light,

chemiluminescence, thus the CL measurements are positively correlated with the amount of

ROS generated15. This study measured the time-course changes in ROS produced during the

oxidative burst in tear-film PMNs stimulated with fMLP, LPS and PMA. We also compared

CL responses between tear-film and blood-isolated PMNs to determine if tear-film PMNs

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have an impaired or reduced ability to initiate a respiratory burst. Both fMLP and LPS bind

to the PMNs cell membrane via receptors, the seven transmembrane spanning G-protein

linked receptors and toll-like receptors (TLR), respectively, to induce different cell functional

responses1,16. On the other hand, PMA diffuses into cells and induces activation by binding

directly to PKC17. These different actions of stimuli may result in the distinct CL responses

observed.

fMLP acts via the pertussis-toxin G-coupled protein to activate several intracellular

signalling pathways involving phospholipase A2 (PLA2), phospholipase C (PLC), mitogen-

activated protein kinase (MAPk), and phosphatidylinositol 3-kinase (PI 3-k) to trigger

various cell responses1,17,18. It has been shown that different functional responses are also

associated with concentrations of stimuli. fMLP at less than 10 nM can only induce

migration, while fMLP at a concentration greater than 100 nM can trigger the respiratory

burst1. In our study, with a stimulus of 1.5µM of fMLP, both tear-film and blood-isolated

PMNs exhibited a peak in ROS production within 3 minutes. Previous work demonstrated

that extracellular ROS were released from PMNs during the first five minutes after

stimulation, while the later CL response is associated mostly with intracellular

production19,20. It is also known that the initial ROS released upon activation by fMLP is

superoxide anion11,15. Our results thus suggest that tear film PMNs are able to produce

extracellular superoxide indicating that the NADPH oxidase on their membrane is functional.

fMLP uses various intracellular pathways to induce the respiratory burst and produce ROS

intra and extracellularly (refer to figure 3 in chapter 2). A study has shown that fMLP-

induced extracellular superoxide production is associated with the extracellular signal-

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regulated kinase (ERK) and phospholipase D (PLD) and is dependent on the MAP kinase

pathway21,22. A rapid and transient activation of MAPk within 1-2 min, which can last for up

to 10 minutes, has also been observed in PMNs23. Hence, the peak and transient generation

of ROS in tear film PMNs suggests that binding of fMLP to its receptor was able to stimulate

PLD through the MAP kinase pathway and result in extracellular production of superoxide.

The extracellular release of superoxide by fMLP-stimulated tear-film PMNs, however, will

need to be further confirmed by using superoxide dismutase (SOD), a known ROS

scavenger11, as these studies have been performed in blood-isolated PMNs.

Blood-isolated PMNs reached a second CL peak around 25 minutes but tear-film

PMNs failed to generate ROS again. The second peak has been associated with intracellular

ROS production19,20 and thus the inability of tear film PMNs to produce intracellular ROS

suggests that some of intracellular pathways leading to ROS may be dysfunctional or

blocked. Our results show that tear-film PMNs can generate ROS under PMA-stimulated

condition so the pathway downstream PKC must be functional. If this is the case, then two

hypotheses can be made around the impairment of intracellular ROS generation with fMLP:

alterations in the downstream signalling/signal transduction from the fMLP receptors or

reduced pool of intracellular signalling molecules. To produce extracellular ROS, the fMLP

receptor must be functional as upon fMLP binding, a Gi signalling pathway is induced to

activate PLD. Thus, the latter hypothesis may be more likely. The intracellular pathways of

fMLP stimulation involve several phospholipases, for example, the PLC which hydrolyzes

phosphatidylinositol into diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), the

latter can increase the intracellular Ca2+ concentration, and DAG serves as a PKC

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activator1,18. The activation of these phospholipases is not only induced by fMLP but also

other inflammatory molecules , such C5a and PAF1. The level of C5a increases dramatically

during sleep24 and thus C5a will bind to its receptor on tear film PMNs and lead to

consumption of intracellular signalling molecules. Collectively, our results may thus suggest

that most of the required substrates for phospholipases have probably already been

hydrolyzed during sleep, and the lack of substrates affect the ability of phospholipases,

resulting in an impaired ability to generate ROS using an intracellular pathway upstream of

PKC. The results with LPS would support such a hypothesis, as upon binding to its receptor

on the cell membrane, it activates similar intracellular signalling pathways. LPS was unable

to trigger the respiratory burst in tear-film PMNs, suggesting that the intracellular

mechanisms are affected. It is possible that the signal transduction from the LPS receptor is

affected but it would be surprising to have the LPS receptor affected and not the fMLP while

they have similar transmembrane receptor characteristics.

PMA was able to induce the respiratory burst in tear-film PMNs, suggesting that the

mechanisms to generate ROS is functional in tear-film PMNs. However, two groups of

participants were observed under PMA-stimulated conditions, the HR and the LR groups,

where the HR produced significantly more ROS than the LR. Similar differences in the

magnitude of ROS response have been observed in blood-isolated PMNs (including our own

study, data not shown) and in PMNs collected from the oral cavities of patients previously

diagnosed with refractory periodontitis25. The participants recruited in this study were

healthy and free of any inflammatory or ocular diseases, suggesting that the higher ROS

production in HR was not due to an underlying inflammatory condition. It has been shown

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that leukocytes express a surface charge, and a lower surface charge is correlated with

increased functional activity26. Hence, HR may possess more PMNs with a high

electronegative charge that are sensitive and hyperactive to stimuli26. PMNs that have been

primed also show an enhanced generation of ROS1. Pro-inflammatory cytokines are present

in the closed-eye environment4, which could prime the tear-film PMNs. It is thus possible

that the higher response observed in the HR may be due to an increased concentration of

inflammatory mediators in the closed-eye environment compared to that of the LR. It is

currently impossible to determine which hypothesis explains the difference in ROS

generation between LR and HR. This warrants further investigation, as the difference in LR

and HR may result in a different ability to respond to bacteria invasion or the presence of a

contact lens and contribute to microbial keratitis and sterile infiltrates.

In a previous study using dihydrorhodamine 123 to measure the respiratory burst via

flow cytometry, it was reported that PMA-stimulated tear-film PMNs had a limited ability to

produce ROS as compared to blood-isolated PMNs3,24, an observation that our current results

contradict. It is evident from our CL study that ROS production is time-dependent. Flow

cytometry, however, can only assess ROS production at one endpoint, which may not be

sufficient to infer the overall ability of ROS generation by tear-film PMNs depending on the

time point chosen. In addition, dihydrorhodamine 123 in flow cytometry measures the

intracellular ROS27, whereas the luminol-enhanced chemiluminescence detects both the

extra- and intracellular ROS generation15. PMA is recognized to stimulate both intra-and

extracellular release of ROS9, thus flow cytometry might underestimate ROS production. To

activate cells, PMA needs to diffuse into the cell and then bind to PKC. The slower

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responding time of tear-film PMNs might be due to difference in cell membrane properties,

resulting in slower diffusion into the cells compared to blood-isolated PMNs, or there might

also be a rate limiting factor in the intracellular pathway, whereby tear film PMNs may

possess lower intracellular concentrations of one or more molecules involved in ROS

generation. Such a hypothesis would be supported by the results with fMPL and LPS

impaired ROS production as discussed above.

Our results also indicated that unstimulated tear film PMNs had a high level of ROS

compared to blood isolated PMNs. The closed eye environment contains several

inflammatory cytokines4 and thus it is not surprising that tear film PMNs may have produced

ROS intracellularly upon exposure to IL-8, C5a and other inflammatory mediators present. It

is currently assumed that the ROS was intracellular as the presence of the intracellular pool

of ROS would allow the tear film PMNs to kill the pathogens that have been phagocytosed

and thus limit potential damage to the ocular surface from releasing ROS. Further

investigation will be necessary to confirm that the measured CL was indeed from

intracellular ROS. However previous flow cytometry results would suggest that this is the

case3.

6.5. Conclusions

Using the luminol-enhanced chemiluminescence assay, we demonstrated that tear-

film PMNs collected from the closed-eye environment were able to produce ROS under

certain conditions. ROS production was stimulus and time dependent, highlighting the

importance of a kinetic study, especially with tear film PMNs, which have a different

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phenotype from blood PMNs and thus may not be able to mount an oxidative response within

the same time period. fMLP induced extracellular ROS in tear film PMNs, suggesting a

functional NADPH oxidase, PLD and MAPk pathway. However, both fMLP and LPS failed

to produce ROS intracellularly in tear film neutrophils, which implied that some of the

intracellular signalling pathways of the tear-film PMNs may be impaired or the substrates for

the phospholipases are insufficient. PMA triggered ROS production in tear-film PMNs,

highlighting that any intracellular signalling impairment would then have to be upstream of

the PKC pathway. Furthermore, our study identified two populations in their potential to

generate ROS: the high responders (HR) and the low responders (LR), whereby the HRs

produced significantly more ROS than the LRs upon PMA stimulation. Further work will be

required to confirm this finding as it has important clinical implications for microbial

keratitis and contact lens-induced infiltrates. The higher levels of initial ROS in unstimulated

samples and failure to mount an oxidative response to LPS and fMLP also provide further

evidence that tear film PMNs collected after sleep have undergone prior activation in the

closed eye environment.

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Chapter 7

Conclusions and Future Work

7.1. Conclusions

The presence in the closed-eye environment and the distinct phenotype of tear-film

PMNs has drawn scientists’ attention to their potential role in the ocular environment as well

as in ocular inflammation. This thesis focused on two aspects, 1) investigating appropriate

experimental procedures to process tear-film PMNs; and 2) assessing the inflammatory

response of tear-film PMNs in terms of expression of surface receptors (when stimulated

with IL-8 and PMA) and the production of reactive oxygen species (ROS) (when stimulated

with fMLP, LPS and PMA).

We demonstrated that centrifugation, fixation, and incubation affected the expression

of cell activation membrane receptors, CD11b, CD16, CD45, CD55 and CD66b differently.

The observed changes may be due to the properties of the receptors and/or their interactions

with the fluorochrome-labelled antibodies. Our results indicated that any additional

centrifugation step following initial cell concentration and long incubation times should be

avoided as they appeared to damage cells leading to receptor internalization. Post-fixation

staining ought to be used with these cells as it led to minimal changes in the expression of

surface receptors compared to unfixed samples. Our results may not generalize to all

membrane receptors but highlight the importance of assessing the impact of experimental

procedures on the expression of surface receptors before performing experiments with tear-

film PMNs to ensure that reliable results are obtained.

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We proved that the gentle eye wash method is currently the most effective in

collecting tear-film PMNs from the ocular surface compared to the patch-OSCCA collection

method. While the numbers of total cells collected using the gentle eye wash method were

not consistent across the collection days (day 29 showed significantly fewer cells than day 2),

our study indicated that participants could collect cells well within one day of training and

that the expression of membrane receptors CD11b, CD45 and CD66b was consistent across

collections. However, significant variabilities within a participant as well as within our

population were observed with CD16 and CD55, indicating that CD16 and CD55 may not be

reliable markers to use for diagnostic purposes.

We determined that tear-film PMNs were unable to alter their surface receptors when

stimulated with IL-8, but that they had a limited potential to modulate their expression of

surface receptors when exposed to PMA. A similar response trend was observed with the

respiratory burst whereby tear-film PMNs could generate ROS in response to PMA, but not

fMLP and LPS. Unlike PMA, IL-8, fMLP, and LPS stimulation act via a receptor

transduction signalling process, thus our results suggest that the inability of tear-film PMNs

to induce an inflammatory response is more related to dysfunctional receptors and/or the

impairment of intracellular signalling pathways that are upstream of the PKC pathway.

In this research, we also identified that unstimulated tear-film PMNs displayed a high

expression of CD11b, CD66b and CD55, low expression of CD16, and a high level of ROS

which suggest that they have already been activated in the closed-eye environment.

Taken all together, this thesis contributed significant new knowledge to the

characteristics of tear-film PMNs. Our study demonstrated that tear-film PMNs are sensitive

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to preparation procedures, so any potential impact on the expression of surface receptors

needs to be taken into consideration to increase the reproducibility and reliability of results.

Furthermore, tear-film PMNs showed some functionality upon stimulation with PMA

suggesting that biological factors, such as impaired intracellular signalling pathways,

dysfunctional receptors or prolonged exposure to inflammatory mediators in vivo, is

contributing to the reduced or limited inflammatory responses observed in tear-film PMNs

collected from the closed-eye environment. Furthermore, among all the surface receptors,

while interesting from an inflammatory perspective, CD16 may not be a reliable marker with

tear-film PMNs due to its low level of expression, its hypersensitivity to experimental

procedures, and its inconsistency in the expression across the collection days. While many

questions remain to be answered, this thesis has allowed us to gain a better understanding of

tear-film PMNs and will support future studies to investigate their phenotype and implication

in ocular health and inflammation.

7.2. Future work

We presented some findings of the tear-film PMNs regarding the sensitivity to

experimental procedures, the responses to IL-8, and the ability to generate ROS, but further

research is needed to characterize the phenotype of tear film PMNs as well as conduct

clinical studies to determine their role in ocular infection and inflammation. The first

sections of the future work is related to phenotype the tear-film PMNs isolated from healthy

people to study their functionality, origin and also the possible mechanisms leading to the

noninflammatory phenotype (7.2.1 to 7.2.5) while the remainder focusses on investigation of

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tear-film PMNs obtained from people with ocular inflammation and lens wearers.

7.2.1 Further characterization of tear-film PMNs phenotype – Granules in tear film

PMNs

In Chapter 5, we demonstrated that PMA could slightly upregulate CD11b, CD55,

and CD66b, and downregulate CD16, indicating tear-film PMNs have limited potential to

respond to PMA. We hypothesized that the reduced ability for tear-film PMNs might due to

the previous release of granules in the closed-eye environment because most of the CD11b is

stored in primary granules and CD55 is stored in secretory vesicles. There is limited

knowledge on the granule content of tear film PMNs as all the studies so far have indirectly

measured granule release. There is thus a need to better characterize granules in tear film

PMNs. Imaging flow cytometry presents a great opportunity to visualize the granules within

the tear-film PMNs and assess heterogeneity within a population1,2. Granule staining

combined with surface receptor staining would allow to gain significant insights to determine

if the inability of tear-film PMNs to modulate their expression of surface receptors is due to

the previous activation in the closed-eye environment and release of granules.

7.2.2 Further characterization of tear-film PMNs phenotype – Apoptosis

In addition, it has been shown that the activation of p38 kinases3 and the

downregulation of CD164 are associated with cell apoptosis. If cells are undergoing

apoptosis, mechanisms of cell activation would be affected and thus may explain some of our

results. While tear-film PMNs were found not to be apoptotic using caspase activation5,

further experiments should be performed to determine if they are at an early stage of

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apoptosis when collected, with Annexin V, for example.

7.2.3 Further characterization of tear-film PMNs phenotype – Cytokine release

PMNs can secrete various proinflammatory cytokines, such as interleukin 8 (IL-8),

tumour necrosis factor / (TNF-/), granulocyte-colony stimulating factor (G-CSF) and

interleukin 6 (IL-6)6, in response to stimulus. The primary function of these cytokines is to

prime PMNs in order to amplify their inflammatory response and act as a feedback loop on

cell activation. Our findings demonstrated that tear-film PMNs cannot generate ROS in

response to LPS and fMLP, so it would be essential to test if tear-film PMNs can produce

and release cytokines upon stimulation, as it would help to understand the impairment of

ROS generation in tear-film PMNs.

7.2.4 Further characterization of tear-film PMNs phenotype – Inducing tear film PMNs

phenotype in blood-isolated PMNs with IL-8 exposure

Combining our current results with recent work from Postnikoff & Nichols7, there is

strong evidence that tear film PMNs have been previously activated in the closed eye

environment. Thus, when we assess their phenotype after collection, we are potentially

looking at a second activation stimulus and then comparing their level of activation to blood-

isolated PMNs, which may not be an appropriate reference. Previous work has also tried to

reproduce the phenotype of tear film PMNs and demonstrated that exposure to hypoxic

environment, human corneal epithelial cells and artificial tear solution could not induce the

noninflammatory phenotype in blood-isolated PMNs8. The closed-eye environment has been

shown to contain a high concentration of cytokines that can prime tear-film PMNs and may

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significantly contribute to their distinct phenotype. This has yet to be further explored and

would help us in better understanding the limited response observed with stimulus, such as

LPS, fMLP and IL-8. Experiments where blood-isolated PMNs are primed/activated with

cytokines, such as IL-8, and then activated with a stimulus, would be very valuable to

determine if blood-isolated PMNs can become non-inflammatory/non-responsive as tear-film

PMNs. This would provide significant insights on the mechanisms causing the non-

responsive phenotype of tear-film PMNs.

7.2.5 Further characterization of tear-film PMNs phenotype – Using an in vitro model

to characterize mechanisms involved in tear film PMNs phenotype

The upregulation of CD11b and downregulation of L-selectin is correlated with

extravasation, indicating an activated state of cells. Recent work by Postnikoff & Gorbet8

provided limited insights on how transmigration may affect tear film PMNs phenotype. It

would thus be essential to mimic the transmigration of blood-isolated PMNs to verify if the

later noninflammatory phenotype in tear-film PMNs is caused by transmigration and direct

interaction with the corneal and conjunctival epithelium. Such a hypothesis could be tested

using an in vitro cell culture model, as shown in Figure 31. Apart from this, it also allows us

to study the interaction of the “extravasated” blood-isolated PMNs with the human corneal

epithelial cells (hCECs) in order to have a better understanding of the ocular environment.

The corneal epithelial cells could be substituted with conjunctival epithelial cells or a co-

culture of cell type to gain further insights into mechanisms of activation.

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Figure 31 – An in vitro culture model to mimic the extravasation on blood-isolated PMNs.

7.2.6 The role of tear-film PMNs in seasonal allergy

It has been shown that at awakening, there were twice as many leukocytes collected

from people with dry eye disease (DED) and that a higher ratio of granulocytes to

lymphocytes exited in people with DED9. These results suggest that an inflammatory state

may contribute to recruitment of PMNs. In chapter 6, we reported on one case of a seasonal

allergy participant, showing that the tear-film PMNs isolated from the allergy person

responded differently from the healthy participants. There is thus a need to further

investigate the difference in phenotype between healthy people and people who suffer from

seasonal allergies. This would provide valuable knowledge on how seasonal allergies impact

the ocular environment and the presence of inflammatory cells. This may help in the

development of therapies but would also have potential benefit as a tool to determine the

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impact of therapeutic strategies.

7.2.7 The role of tear-film PMNs in lens-related ocular complications

In our study, we identified two responder groups upon stimulation with PMA: a high

(HR) and low responders (LR). The difference in LR and HR may cause some individuals to

be more susceptible to bacteria, resulting in the microbial keratitis (MK) or to damage the

integrity of corneal epithelium, resulting in sterile infiltrates. Only 8 participants were

involved in that specific experiment and thus it would be valuable to recruit more participants

to determine their response. Not only should non-lens wearers be recruited but we should

also assess the response of lens wearers. The study would have to be designed carefully as

lens material would likely have an effect, but such a study would provide significant

knowledge on ocular health and potential for complications in lens wearers versus non lens

wearers.

7.2.8 The role of tear-film PMNs in lens-related ocular infections – in vivo clinical

studies

While tear-film PMNs have been collected on the ocular surface in various

inflammatory diseases such as dry eye9 and meibomian gland dysfunction10, no study has yet

characterized their phenotype in lens-related infections. While our results are from a small

sample size (n=8), the LR and HR groupings highlight that there could be some significant

underlying factors that may contribute to risks of infections. Studies by Carnt et al11,12 have

focused on the role of cytokines in MK (IL-10 and IL-17) and while PMNs are key players in

infections, little is known on their role and presence in human ocular infections. Being able

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to further study tear film PMNs both from the closed-eye and open-eye environments in

people affected by lens-related MK would have to the potential to contribute significant

knowledge to the field of ocular health and contact lens-induced complications, similarly to

what has been achieved in periodontal health and disease2,13 in the past 10 years.

7.2.9 The role of tear-film PMNs in lens-related ocular infections – in vitro model

Wearing contact lenses during sleep increases the incidence of MK14,15. The presence

of contact lenses and bacteria on the ocular surface, together with the hypoxic environment,

and the need to protect the ocular surface without damaging its integrity leads to a set

complex interaction for tear-film PMNs, which might affect their ability to respond to the

bacteria. An in vitro model with tear-film PMNs, bacteria, a contact lens, and probably

human corneal epithelial cells under the hypoxic condition may provide valuable

information. However, based on our study, short incubation would be required, and this may

limit the value of such a model. If we were able to induce the tear film phenotype with

blood-isolated PMNs in 7.2.5 this would be the most useful model.

It would also be interesting to incubate bacteria with different types of lens materials

to examine if there is a particular material that is preferential for bacteria binding and biofilm

formation and determine how the presence of tear-film PMNs and blood-isolated PMNs and

the release of their various mediators would affect the growth/stability of the biofilm. Our

results clearly indicate that tear film PMNs are activated in vivo and thus it would be highly

relevant to understand how such an activation contributes to fighting invading pathogens and

how subsequent stimulus affect or not the outcomes.

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